Compositions for treating neurological disorders

ABSTRACT

The present invention relates to compositions and methods for the treatment of neurological disorders related to glutamate excitotoxicity and Amyloid β toxicity. More specifically, the present invention relates to novel combinatorial therapies of Multiple Sclerosis, Alzheimer&#39;s disease, Alzheimer&#39;s disease related disorders, Amyotrophic Lateral Sclerosis, Parkinson&#39;s disease, Huntington&#39;s disease, neuropathic pain, alcoholic neuropathy, alcoholism or alcohol withdrawal, or spinal cord injury.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thetreatment of neurological diseases and disorders. More particularly,this invention relates to novel combinatorial therapies for suchdiseases, including Alzheimer's and related diseases, MultipleSclerosis, Amyotrophic Lateral Sclerosis, Parkinson's disease,neuropathies, alcoholism, alcohol withdrawal, Huntington's disease andspinal cord injury.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the prototypic cortical dementiacharacterized by memory deficit together with dysphasia (languagedisorder in which there is an impairment of speech and of comprehensionof speech), dyspraxia (disability to coordinate and perform certainpurposeful movements and gestures in the absence of motor or sensoryimpairments) and agnosia (ability to recognize objects, persons, sounds,shapes, or smells) attributable to involvement of the corticalassociation areas (1-4).

AD is at present the most common cause of dementia. It is clinicallycharacterized by a global decline of cognitive function that progressesslowly and leaves end-stage patients bound to bed, incontinent anddependent on custodial care. Death occurs, on average, 9 years afterdiagnosis (5).

The incidence rate of AD increases dramatically with age. United Nationpopulation projections estimate that the number of people older than 80years will approach 370 million by the year 2050. Currently, it isestimated that 50% of people older than age 85 years are afflicted withAD. Therefore, more than 100 million people worldwide will suffer fromdementia in 50 years. The vast number of people requiring constant careand other services will severely affect medical, monetary and humanresources (6). Memory impairment is the early feature of the disease andinvolves episodic memory (memory for day-today events). Semantic memory(memory for verbal and visual meaning) is involved later in the disease.The pathological hallmark of AD includes amyloid plaques containingbeta-amyloid (Abeta), neurofibrillary tangles (NFT) containing Tau andneuronal and synaptic dysfunction and loss (7-9). For the last decade,two major hypotheses on the cause of AD have been proposed: the “amyloidcascade hypothesis”, which states that the neurodegenerative process isa series of events triggered by the abnormal processing of the AmyloidPrecursor Protein (APP) (10), and the “neuronal cytoskeletaldegeneration hypothesis” (11), which proposes that cytoskeletal changesare the triggering events. The most widely accepted theory explaining ADprogression remains the amyloid cascade hypothesis (12-14) and ADresearchers have mainly focused on determining the mechanisms underlyingthe toxicity associated with Abeta proteins. Microvascular permeabilityand remodeling, aberrant angiogenesis and blood brain barrier breakdownhave been identified as key events contributing to the APP toxicity inthe amyloid cascade (15). On contrary, Tau protein has received muchless attention from the pharmaceutical industry than amyloid, because ofboth fundamental and practical concerns. Moreover, synaptic densitychange is the pathological lesion that best correlates with cognitiveimpairment than the two others.

Studies have revealed that the amyloid pathology appears to progress ina neurotransmitter-specific manner where the cholinergic terminalsappear most vulnerable, followed by the glutamatergic terminals andfinally by the GABAergic terminals (9). Glutamate is the most abundantexcitatory neurotransmitter in the mammalian nervous system. Underpathological conditions, its abnormal accumulation in the synaptic cleftleads to glutamate receptors overactivation (16). Abnormal accumulationof glutamate in synaptic cleft leads to the overactivation of glutamatereceptors that results in pathological processes and finally in neuronalcell death. This process, named excitotoxicity, is commonly observed inneuronal tissues during acute and chronic neurological disorders.

It is becoming evident that excitotoxicity is involved in thepathogenesis of multiple disorders of various etiology such as: spinalcord injury, stroke, traumatic brain injury, hearing loss, alcoholismand alcohol withdrawal, alcoholic neuropathy, or neuropathic pain aswell as neurodegenerative diseases such as multiple sclerosis,Alzheimer's disease, Amyotrophic Lateral Sclerosis, Parkinson's disease,and Huntington's disease (17-19). The development of efficient treatmentfor these diseases remains major public health issues due to theirincidence as well as lack of curative treatments.

NMDAR antagonists that target various sites of this receptor have beentested to counteract excitotoxicity. Uncompetitive NMDAR antagoniststarget the ion channel pore thus reducing the calcium entry intopostsynaptic neurons. Some of them reached the approval status. As anexample, Memantine is currently approved in moderate to severeAlzheimer's disease. It is clinically tested in other indications thatinclude a component of excitotoxicity such as alcohol dependence (phaseII), amyotrophic lateral sclerosis (phase III), dementia associated withParkinson (Phase II), epilepsy, Huntington's disease (phase IV),multiple sclerosis (phase IV), Parkinson's disease (phase IV) andtraumatic brain injury (phase IV). This molecule is however of limitedbenefit to most Alzheimer's disease patients, because it has only modestsymptomatic effects. Another approach in limiting excitotoxicityconsists in inhibiting the presynaptic release of glutamate. Riluzole,currently approved in amyotrophic lateral sclerosis, showed encouragingresults in ischemia and traumatic brain injury models (20-23). It is atpresent tested in phase II trials in early multiple sclerosis,Parkinson's disease (does not show any better results than placebo) aswelt as spinal cord injury. In 1995, the drug reached orphan drug statusfor the treatment of amyotrophic lateral sclerosis and in 1996 for thetreatment of Huntington's disease.

WO2009/133128, WO2009/133141, WO2009/133142, and WO2011/054759, disclosemolecules which can be used in compositions for treating neurologicaldisorders.

Despite active research in this area, there is still a need foralternative or improved efficient therapies for neurological disorders,and, in particular, neurological disorders which are related toglutamate and/or amyloid beta toxicity. The present invention providesnew treatments for such neurological diseases of the central nervoussystem (CNS) and the peripheral nervous system (PNS).

SUMMARY OF INVENTION

An object of the present invention is to provide new therapeuticapproaches for treating neurological disorders.

The invention stems, inter alia, from the unexpected discovery by theinventors that Torasemide, Trimetazidine, Mexiletine, Bromocriptine,Ifenprodil and Moxifloxacin, alone or in combinations, represent new andeffective therapies for the treatment of neurological disorders.

The invention therefore provides novel compositions and methods fortreating neurological disorders, particularly AD and related disorders,Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS),Parkinson's disease (PD), neuropathies (for instance neuropathic pain oralcoholic neuropathy), alcoholism or alcohol withdrawal, damages to theperipheral nervous system, Huntington's disease (HD) and spinal cordinjury.

More particularly, the invention relates to a composition, for use inthe treatment of a neurological disorder, comprising at leastTorasemide, Trimetazidine, Mexiletine, Ifenprodil, Moxifloxacin orBromocriptine, or a salt, prodrug, derivative, or sustained releaseformulation thereof.

A further object of the present invention relates to a compositioncomprising at least one first compound selected from the groupconsisting of Torasemide, Trimetazidine, Mexiletine, Ifenprodil,Moxifloxacin, and Bromocriptine, or a salt, prodrug, derivative of anychemical purity, or sustained release formulation thereof, incombination with at least one second compound distinct from said firstcompound, selected from Sulfisoxazole, Methimazole, Priocaine,Dyphylline, Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid,Baclofen, Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine,Eplerenonc, Fenoldopam, Leflunomide, Levosimendan, Sulodexide,Terbinafine, Zonisamide, Etomidate, Phenformin, Trimetazidine,Mexiletine, Bromocriptine, Ifenprodil, Torasemide, and Moxifloxacinsalts, prodrugs, derivatives of any chemical purity, or sustainedrelease formulation thereof, for simultaneous, separate or sequentialadministration.

A further object of the present invention relates to a composition, foruse in the treatment of a neurological disorder, comprising at least onefirst compound selected from the group consisting of Torasemide,Trimetazidine, Mexiletine, Ifenprodil, Moxifloxacin, and Bromocriptine,salts, prodrugs, derivatives of any chemical purity, or sustainedrelease formulation thereof, in combination with at least one secondcompound distinct from said first compound, selected from Sulfisoxazole,Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone,Acamprosate, Aminocaproic acid, Baclofen, Cabergoline,Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam,Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide,Etomidate, Phenformin, Trimetazidine, Mexiletine, Bromocriptine,Ifenprodil, Torasemide, and Moxifloxacin, salts, prodrugs, derivativesof any chemical purity, or sustained release formulation thereof, forsimultaneous, separate or sequential administration.

The present invention also relates to a composition comprising at leastone first compound selected from the group consisting of Torasemide,Trimetazidine, Mexiletine, Ifenprodil, Moxifloxacin and Bromocriptine,salt(s), prodrug(s), derivative(s) of any chemical purity, or sustainedrelease formulation(s) thereof, in combination with at least one secondcompound distinct from said first compound, selected from Sulfisoxazole,Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone,Acamprosate, Aminocaproic acid, Baclofen, Cabergoline,Diethylcarbamazine, Cinacalcet Cinnarizine, Eplerenone, Fenoldopam,Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide,Etomidate, Phenformin, Trimetazidine, Mexiletine, Bromocriptine,Ifenprodil, Torasemide, and Moxifloxacin salt(s), prodrug(s),derivative(s) of any chemical purity, or sustained releaseformulation(s) thereof, and a pharmaceutically acceptable excipient, forsimultaneous, separate or sequential administration.

Most preferred drug compositions comprise 1, 2, 3, 4 or 5 distinctdrugs, even more preferably 2, 3 or 4. Furthermore, the above drugcompositions may also be used in further combination with one or severaladditional drugs or treatments beneficial to subjects with aneurological disorder.

The invention also relates to a method of treating a neurologicaldisorder, the method comprising administering to a subject in needthereof a drug or composition as disclosed above.

A further object of this invention relates to a method of treating aneurological disorder, the method comprising simultaneously, separatelyor sequentially administering to a subject in need thereof a drugcombination as disclosed above.

A further object of this invention relates to the use of at least onecompound selected from the group consisting of Torasemide,Trimetazidine, Mexiletine, Ifenprodil, Bromocriptine and Moxifloxacin,or salt(s), prodrug(s), derivative(s) of any chemical purity, orsustained release formulation(s) thereof, for the manufacture of amedicament for the treatment of a neurological disorder.

A further object of this invention relates to the use of drugcombinations disclosed above, for the manufacture of a medicament forthe treatment of a neurological disorder.

The invention may be used in any mammalian subject, particularly humansubject, at any stage of the disease.

BRIEF DESCRIPTION OF THE FIGURES

For FIGS. 1 to 27, *: p<0.05: significantly different from control (nointoxication); “ns”: no significant effect (ANOVA+Dunnett's Post-Hoctest).

FIGS. 1A-1C: Effect of selected drugs pre-treatment against human Aβ₁₋₄₂injury in HBMEC. A) Validation of the experimental model used for drugscreening: 1 hr of VEGF pre-treatment at 10 nM significantly protectedthe capillary network from this amyloid injury (+70% of capillarynetwork compared to amyloid intoxication). The intoxication issignificantly prevented by Torasemide (B) and Bromocriptine (C) at dosesas low as of 400 nM and 3.2 nM respectively, whereas no or a weakereffect is noticed for upper and lower doses.

: p<0.05: significantly different from Amyloid intoxication.

FIGS. 2A-2F: Effect of selected drugs pre-treatment on LDH release inhuman Aβ₁₋₄₂ toxicity assays on rat primary cortical cells. A)Validation of the experimental model used for drug screening: 1 hr ofEstradiol (150 ng/ml) pre-treatment significantly protected the neuronsfrom this amyloid injury (−70%), which is considered as a positivecontrol for neuroprotection. For all experiments, Aβ₁₋₄₂ produces asignificant intoxication compared to vehicle-treated neurons. Theintoxication is significantly prevented by Bromocriptine (40 nM, −29%)(B), Trimetazidine (40 nM, −94%) (C), Ifenprodil (600 nM, −94%) (D),Mexiletine (3.2 nM, −73%) (E), Moxifloxacin (20 nM, −63%) (F). Note thatfor other drug concentrations, no or a weaker effect is noticed forupper and lower doses.

: p<0.05: significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 3A-3C: Effect of Baclofen and Torasemide combination therapy onthe total length of capillary network in beta-amyloid intoxicated HBMECcultures. The aggregated human amyloid peptide (Aβ₁₋₄₂ 2.5 μM) producesa significant intoxication, above 40%, compared to vehicle-treatedcells. This intoxication is significantly prevented by the combinationof Baclofen and Torasemide (A) whereas, at those concentrations,Baclofen (B) and Torasemide (C) alone have no significant effect onintoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 4A-4C: Effect of Sulfisoxazole and Torasemide combination therapyon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures. The aggregated human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 40%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of Sulfisoxazole and Torasemide (A) whereas, at thoseconcentrations, Sulfisoxazole (B) and Torasemide (C) alone have nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 5A-5C: Effect of Eplerenone and Torasemide combination therapy onthe total length of capillary network in beta-amyloid intoxicated HBMECcultures. The aggregated human amyloid peptide (Aβ₁₋₄₂ 2.5 μM) producesa significant intoxication, above 40%, compared to vehicle-treatedcells. This intoxication is significantly prevented by the combinationof Eplerenone and Torasemide (A) whereas, at those concentrations,Torasemide (B) and Eplerenone (C) alone have no significant effect onintoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 6A-6C: Effect of Bromocriptine and Sulfisoxazole combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures. The aggregated human amyloid peptide (Aβ₁₋₄₂2.5 μM) produces a significant intoxication, above 40%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of Bromocriptine and Sulfisoxazole (A) whereas, at thoseconcentrations, Bromocriptine (B) and Sulfisoxazole (C) alone have nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 7A-7C: Effect of Acamprosate and Ifenprodil combination therapy onLDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells. Theaggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces a significantintoxication compared to vehicle-treated neurons. This intoxication issignificantly prevented by the combination of Acamprosate and Ifenprodil(A) whereas, at those concentrations, Acamprosate (B) and Ifenprodil (C)alone have no significant effect on intoxication,

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 8A-8C: Effect of Baclofen and Mexiletine combination therapy onLDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells. Theaggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces a significantintoxication compared to vehicle-treated neurons. This intoxication issignificantly prevented by the combination of Baclofen and Mexiletine(A) whereas, at those concentrations, Baclofen (B) and Mexiletine (C)alone have no significant effect on intoxication,

: p=0.051, different from Aβ₁₋₄₂, intoxication.

FIGS. 9A-9C: Effect of Baclofen and Trimetazidine combination therapy onLDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells. Theaggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces a significantintoxication compared to vehicle-treated neurons. This intoxication issignificantly prevented by the combination of Baclofen and Trimetazidine(A) whereas, at those concentrations, Baclofen (B) and Trimetazidine (C)alone have no significant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 10A-10C: Effect of Cinacalcet and Mexiletine combination therapyon LDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells.The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination of Cinacalcetand Mexiletine (A) whereas, at those concentrations, Cinacalcet (B) andMexiletine (C) alone have no significant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 11A-11C: Effect of Cinnarizine and Trimetazidine combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofCinnarizine and Trimetazidine (A) whereas, at those concentrations,Cinnarizine (B) and Trimetazidine (C) alone have no significant effecton intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 12A-12C: Effect of Trimetazidine and Zonisamide combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofTrimetazidine and Zonisamide (A) whereas, at those concentrations,Trimetazidine (B) and Zonisamide (C) alone have no significant effect onintoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 13A-13C: Effect of Terbinafine and Torasemide combination therapyon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures. The aggregated human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 40%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of Terbinafine and Torasemide (A) whereas, at thoseconcentrations, Terbinafine (B) and Torasemide (C) alone have nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 14A-14C: Effect of Cinacalcet and Mexiletine combination therapyon the total length of capillary network in beta-amyloid intoxicatedHBMEC cultures. The aggregated human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 40%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of Cinacalcet and Mexiletine (A) whereas, at thoseconcentrations, Cinacalcet (B) and Mexiletine (C) alone have nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIG. 15: Effect of Baclofen and Torasemide combination therapy on LDHrelease in human Aβ₁₋₄₂ toxicity on rat primary cortical cells. Theaggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces a significantintoxication compared to vehicle-treated neurons. This intoxication issignificantly prevented by the combination of Baclofen and Torasemidewhereas, at those concentrations, Baclofen and Torasemide alone have nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 16A-16C: Effect of Torasemide and Sulfisoxazole combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofSulfisoxazole and Torasemide (A) whereas, at those concentrations,Torasemide (B) and Sulfisoxazole (C) alone have no significant effect onintoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 17A-17C: Effect of Moxifloxacin and Trimetazidine combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofMoxifloxacin and Trimetazidine (A). Adjunction of Moxifloxacin allows anincrease of 100% of the effect observed for Trimetazidine (C) alone,whereas, at the same concentration, Moxifloxacin (9) alone has nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 18A-18C: Effect of Moxifloxacin and Baclofen combination therapyon LDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells.The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofMoxifloxacin and Baclofen (A) whereas, at those concentrations,Moxifloxacin (B) and Baclofen (C) alone have no significant effect onintoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 19A-19C: Effect of Moxifloxacin and Cinacalcet combination therapyon LDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells.The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofMoxifloxacin and Cinacalcet (A) whereas, at those concentrations,Moxifloxacin (B) and Cinacalcet (C) alone have no significant effect onintoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 29A-20C: Effect of Moxifloxacin and Zonisamide combination therapyon LDH release in human Aβ₁₋₄₂ toxicity on rat primary cortical cells.The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofMoxifloxacin and Zonisamide (A). Adjunction of Moxifloxacin allows anincrease of 81% of the effect observed for Zonisamide (C) alone,whereas, at the same concentration, Moxifloxacin (B) alone has nosignificant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 21A-21C: Effect of Moxifloxacin and Sulfisoxazole combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination ofMoxifloxacin and Sulfisoxazole (A) whereas, at those concentrations,Moxifloxacin (B) and Sulfisoxazole (C) alone have no significant effecton intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIG. 22: Effect of Mexiletine (MEX) and Ifenprodil (IFN) combinationtherapy on LDH release in human Aβ₁₋₄₂, toxicity on rat primary corticalcells. The aggregated human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces asignificant intoxication compared to vehicle-treated neurons. Thisintoxication is significantly prevented by the combination of Mexiletine25.6 pM and Ifenprodil 24 nM whereas, at those concentrations,Mexiletine and Ifenprodil alone have no significant effect onintoxication.

: p<0.0572, significantly different from Aβ₁₋₄₂ intoxication.

FIG. 23: Effect of Baclofen (BCL) and Torasemide (TOR) combinationtherapy on the total length of neurites network in beta-amyloidintoxicated cortical neurons. The human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 15%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of Torasemide and Baclofen; furthermore this combinationallows an enhancement of neurite growth.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIG. 24: Effect of Cinacalcet and Mexiletine combination therapy againstglutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination of Cinacalcet(64 pM) and Mexiletine (25.6 pM) whereas, at those concentrations,Cinacalcet and Mexiletine alone have no significant effect onintoxication.

: p<0.001, significantly different from glutamate intoxication;(ANOVA+Dunnett Post-Hoc test).

FIG. 25: Effect of Sulfisoxazole and Torasemide combination therapyagainst glutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination ofSulfisoxazole (6.8 nM) and Torasemide (400 nM) whereas, at thoseconcentrations, Sulfisoxazole and Torasemide alone have no significanteffect on intoxication.

: p<0.001; significantly different from glutamate intoxication;(ANOVA+Dunnett Post-Hoc test).

FIG. 26: Effect of Torasemide (TOR) pre-treatment on LDH release inhuman Aβ₁₋₄₂ toxicity assays on rat primary cortical cells. Aβ₁₋₄₂produces a significant intoxication compared to vehicle-treated neurons.The intoxication is significantly prevented by Torasemide (200 nM,−90%). ⋄: p<0.0001: significantly different from Aβ₁₋₄₂ intoxication.

FIG. 27: Comparison of Acamprosate and its derivative Homotaurinepre-treatment on LDH release in human Aβ₁₋₄₂ toxicity assays on ratprimary cortical cells. Aβ₁₋₄₂ produces a significant intoxicationcompared to vehicle-treated neurons. The intoxication is equallysignificantly prevented by Homotaurine and Acamprosate (99%, 8 nM). ⋄:p<0.0001: significantly different from Aβ₁₋₄₂ intoxication.

FIG. 28: Effect of Baclofen (BCL) and Torasemide (TOR) combination onthe total length of neurites network of cortical neurons cultured inabsence of toxic. An increase of neurite network length is observed whenBaclofen (400 nM) and Torasemide (80 nM) combination is added in theculture medium; furthermore this combination allows an enhancement ofneurite growth whereas, at those concentrations, Baclofen and Torasemidealone have no significant (ns) effect on neurite network length. *p<0.005, significantly different from control.

FIGS. 29A-29B: Effect of Baclofen (BCL) and Torasemide (TOR) combinationin promoting nerve regeneration after nerve crush. A—Animalsexperiencing nerve injury (nerve crush) treated with baclofen-torasemidecombination show a significantly lower latency of CMAP upon stimulationof the injured sciatic nerve at day 7 and day 30 from nerve crush whencompared to the sham operated animals (white bar) or to the vehicletreated animals. B—Amplitudes of the signal of muscular evokedpotentials upon sciatic nerve stimulation are significantly lower inanimals experiencing nerve injury when compared to the sham operatedanimals at both day 7 or day 30 from nerve crush. A significant increasein CMAP amplitude is observed at day 30 from nerve crush for the animalstreated with baclofen (3 mg/kg)-torasemide (400 μg/kg) (dose 3) bid.*p<0.05; **p<0.001; ***p<0.0001, significantly different from vehicletreated animals (black bar).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new compositions for treatingneurological disorders. The invention discloses novel use of drugs ornovel drug combinations which allow an effective correction of suchdiseases and may be used for patient treatment.

The invention is suited for treating any neurological disorder, whethercentral or peripheral, particularly disorders wherein amyloid orglutamate excitotoxicity are involved. Specific examples of suchdisorders include neurodegenerative diseases such as Alzheimer's andrelated disorders, Multiple Sclerosis (MS), Amyotrophic LateralSclerosis (ALS), Parkinson's Disease (PD), Huntington's Disease (HD), orother neurological disorders like neuropathies (for instance alcoholicneuropathy or neuropathic pain), alcoholism or alcohol withdrawal andspinal cord injury. “Neuropathies” refers to conditions where nerves ofthe peripheral nervous system are damaged, this include damages of theperipheral nervous system provoked by genetic factors, inflammatorydisease, by chemical substance including drugs (vincristine,oxaliplatin, ethyl alcohol), or by a direct physical insult to thenerve. The treatment of neuropathies also includes the treatment ofneuropathic pain.

Damages of the peripheral nervous system can be ranked according to thestage of neuronal insult. Invention is suited for treating nerveinjuries ranking from neurapraxia (condition where only signallingability of the nerve is impaired), axonometsis (injury implying damagesto the axons, without impairing surrounding connective tissues of thenerves), but also neurotmesis (injury damaging both axon and surroundingtissues).

Alterations in axon or in surrounding tissues as myelin) can be ofgenetic origin. An example of inherited neuropathies is the so calledCharcot Marie Tooth family of diseases. Charcot Marie Tooth diseases areprogressive disorders that affect peripheral nerves which aredistinguished by the specific gene(s) that is (are) altered, Mutation(s)result in an impairment of axons, which transmit nerve impulses, and/oraffect the production myelin sheath by Shwann cells, which is implicatedin the speed of the transmission of nervous impulse. There are severaltypes (categorized as a function of clinical features) and subtypes ofCMT (corresponding to a genetic classification). CMT types are CMT1,CMT2, CMT3, CMT/1, CMT5, CMT6, CMTDI, CMTRI, CMTX. Related peripheralneuropathies are, for example, HNPP (hereditary neuropathy withliability to pressure palsies), severe demyelinating neuropathies DSS(Dejerine-Sottas syndrome), GIN (congenital hypomyelinating neuropathy).CMT1 (a demyelinating type) and CMT2 (an axonal type) account for around70% of the CMT patients.

The invention is particularly suited for treating AD and relateddisorders. In the context of this invention, the term. “AD relateddisorder” includes senile dementia of AD type (SDAT), Lewis bodydementia, vascular dementia, mild cognitive impairment (MCI) andage-associated memory impairment (AAMI).

As used herein, “treatment” includes the therapy, prevention,prophylaxis, retardation or reduction of symptoms provoked by or of thecauses of the above diseases or disorders. The term treatment includesin particular the control of disease progression and associatedsymptoms. The term treatment particularly includes i) a protectionagainst the toxicity caused by Amyloid Beta, or a reduction orretardation of said toxicity, and/or ii) a protection against glutamateexcitotoxicity, or a reduction or retardation of said toxicity, in thetreated subjects. The term treatment also designates an improvement ofcognitive symptom or a protection of neuronal cells. In relation toneuropathies, the term treatment also includes nerve regeneration, whichencompasses remyelination, generation of new neurons, glia, axons,myelin or synapses.

Within the context of this invention, the designation of specificcompounds is meant to include not only the specifically named molecules,but also any pharmaceutically acceptable salt, hydrate, derivatives(e.g., ester, ether), isomers, racemate, conjugates, or prodrugsthereof, of any purity.

The term “prodrug” as used herein refers to any functional derivatives(or precursors) of a compound of the present invention, which, whenadministered to a biological system, generates said compound as a resultof e.g., spontaneous chemical reaction(s), enzyme catalyzed chemicalreaction(s), and/or metabolic chemical reaction(s). Prodrugs are usuallyinactive or less active than the resulting drug and can be used, forexample, to improve the physicochemical properties of the drug, totarget the drug to a specific tissue, to improve the pharmacokinetic andpharmacodynamic properties of the drug and/or to reduce undesirable sideeffects. Prodrugs typically have the structure X-drug wherein X is aninert carrier moiety and drug is the active compound, wherein theprodrug is less active than the drug and the drug is released from thecarrier in vivo. Some of the common functional groups that are amenableto prodrug design include, but are not limited to, carboxylic, hydroxyl,amine, phosphate/phosphonate and carbonyl groups. Prodrugs typicallyproduced via the modification of these groups include, but are notlimited to, esters, carbonates, carbamates, amides and phosphates.Specific technical guidance for the selection of suitable prodrugs isgeneral common knowledge (24-28). Furthermore, the preparation ofprodrugs may be performed by conventional methods known by those skilledin the art. Methods which can be used to synthesize other prodrugs aredescribed in numerous reviews on the subject (25; 29-35). For example,Arbaclofen Placarbil is listed in ChemID plus Advance database(http://chem.sis.nlm.nih.gov/chemidplus/) and Arbaclofen Placarbil is awell known prodrug of Baclofen (36; 43).

The term “derivative” of a compound includes any molecule that isfunctionally and/or structurally related to said compound, such as anacid, amide, ester, ether, acetylated variant, hydroxylated variant, oran alkylated (C1-C6) variant of such a compound. The term derivativealso includes structurally related compound having lost one or moresubstituent as listed above. For example, Homotaurine is a deacetylatedderivative of Acamprosate. Preferred derivatives of a compound aremolecules having a substantial degree of similarity to said compound, asdetermined by known methods. Similar compounds along with their index ofsimilarity to a parent molecule can be found in numerous databases suchas PubChem (http://pubchem.ncbi.nlm.nih.gov/search/) or DrugBank(http://www.drugbank.ca/). In a more preferred embodiment, derivativesshould have a Tanimoto similarity index greater than 0.4, preferablygreater than 0.5, more preferably greater than 0.6, even more preferablygreater than 0.7 with a parent drug. The Tanimoto similarity index iswidely used to measure the degree of structural similarity between twomolecules. Tanimoto similarity index can be computed by software such asthe Small Molecule Subgraph Detector (37-38) available online(http://www.ebi.ac.uk/thornton-srv/software/SMSD/). Preferredderivatives should be both structurally and functionally related to aparent compound, i.e., they should also retain at least part of theactivity of the parent drug, more preferably they should have aprotective activity against Aβ or glutamate toxicity.

The term derivatives also include metabolites of a drug, e.g., amolecule which results from the (biochemical) modification(s) orprocessing of said drug after administration to an organism, usuallythrough specialized enzymatic systems, and which displays or retains abiological activity of the drug. Metabolites have been disclosed asbeing responsible for much of the therapeutic action of the parent drug.In a specific embodiment, a “metabolite” as used herein designates amodified or processed drug that retains at least part of the activity ofthe parent drug, preferably that has a protective activity against Aβtoxicity or glutamate toxicity. Examples of metabolites includehydroxylated forms of Torasemide resulting from the hepatic metabolismof the drug (Drug bank database (39).

The term “salt” refers to a pharmaceutically acceptable and relativelynon-toxic, inorganic or organic acid addition salt of a compound of thepresent invention. Pharmaceutical salt formation consists in pairing anacidic, basic or zwitterionic, drug molecule with a counterion to createa salt version of the drug. A wide variety of chemical species can beused in neutralization reaction. Pharmaceutically acceptable salts ofthe invention thus include those obtained by reacting the main compound,functioning as a base, with an inorganic or organic acid to form a salt,for example, salts of acetic acid, nitric acid, tartric acid,hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonicacid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid orcitric acid. Pharmaceutically acceptable salts of the invention alsoinclude those in which the main compound functions as an acid and isreacted with an appropriate base to form, e.g., sodium, potassium,calcium, magnesium, ammonium, or choline salts. Though most of salts ofa given active principle are bioequivalents, some may have, amongothers, increased solubility or bioavailability properties. Saltselection is now a common standard operation in the process of drugdevelopment as taught by H. Stahl and C. G Wermuth in their handbook(40).

The term “combination” or “combinatorial treatment/therapy” designates atreatment wherein at least two or more drugs are co-administered to asubject to cause a biological effect. In a combined therapy according tothis invention, the at least two drugs may be administered together orseparately, at the same time or sequentially. Also, the at least twodrugs may be administered through different routes and protocols. As aresult, although they may be formulated together, the drugs of acombination may also be formulated separately.

As disclosed in the examples, Torasemide, Trimetazidine, Mexiletine,Ifenprodil, Bromocriptine and Moxifloxacin have a strong unexpectedeffect on biological processes involved in neurological disorders.Furthermore, these compounds also showed in vivo a very efficientability to correct symptoms of such diseases. These molecules, alone orin combination therapies, therefore represent novel approaches fortreating neurological disorders, such as Alzheimer's disease, MultipleSclerosis, Amyotrophic Lateral Sclerosis, Parkinson's Disease,Huntington's Disease, neuropathies (for instance neuropathic pain oralcoholic neuropathy), alcoholism or alcohol withdrawal, and spinal cordinjury. Combinations of these drugs with other selected compounds (seeTable 2) are particularly advantageous because they produce a surprisingand unexpected synergistic effect at dosages where the drugs alone haveessentially no effect. Also, because of their efficacy, the hereindisclosed drugs combinations can be used at low dosages, which is afurther very substantial advantage.

In this regard, in particular embodiment, the invention relates to acomposition for use in the treatment of AD, AD related disorders, MS,PD, ALS, HD, neuropathies (for instance neuropathic pain or alcoholicneuropathy), alcoholism or alcohol withdrawal, or spinal cord injury,comprising at least Torasemide, Trimetazidine, Mexiletine, Ifenprodil,Bromocriptine, or Moxifloxacin, or a salt, prodrug, derivative, orsustained release formulation thereof.

The specific CAS number for each of these compounds is provided in Table1 below. Table 1 cites also, in a non-limitative way, common salts,racemates, prodrugs, metabolites or derivatives for these compounds usedin the compositions of the invention.

TABLE 1 Class or Tanimoto similarity Drug CAS Numbers index Mexiletineand related compounds Mexiletine 31828-71-4; 5370-01-46-Hydroxymethylmexiletine 53566-98-6 Metabolite 4-Hydroxymexiletine53566-99-7 Metabolite 3-Hydroxymexiletine (MHM) 129417-37-4 MetaboliteN-Hydroxymexiletine 151636-18-9 Metabolite glucuronide Sulfisoxazole andrelated compounds Sulfisoxazole 127-69-5; 4299-60-9N(4)-Acetylsulfisoxazole 4206-74-0 Metabolite Sulfisoxazole acetyl80-74-0 Prodrug Sulfamethoxazole 723-46-6 0.52 Cinacalcet and relatedcompounds Cinacalcet 226256-56-0; 364782-34-3 Hydrocinnamic acid501-52-0 Metabolite Torasemide and related compounds Torasemide56211-40-6; 72810-59-4 Hydroxytorasemide 99300-68-2; 99300-67-1Metabolites Carboxytorasemide Metabolite Tolbutamide 64-77-7 0.55Bromocriptine and related compounds Bromocriptine 25614-03-3; 22260-51-1Ifenprodil and related compounds Ifenprodil 23210-56-2; 23210-58-4

The above molecules may be used alone or, preferably, in combinationtherapies to provide the most efficient clinical benefit. In thisregard, in a preferred embodiment, the invention relates to acomposition for use in the treatment of a neurological disorder,preferably AD, AD related disorders, MS, PD, ALS, HD, neuropathies (forinstance neuropathic pain or alcoholic neuropathy), alcoholism oralcohol withdrawal, or spinal cord injury, comprising any one of theabove compounds in combination with at least one distinct compoundselected from Sulfisoxazole, Methimazole, Prilocaine, Dyphylline,Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen,Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone,Fenoldopam, Leflunomide, Levosimendan, Sulodexide, Terbinafine,Zonisamide, Etomidate, Phenformin, Trimetazidine, Mexiletine,Ifenprodil, Moxifloxacin, Bromocriptine or Torasemide, or a salt,prodrug, derivative, or sustained release formulation thereof.

The specific CAS number for each of these additional distinct compounds,different from those of Table 1 is provided in Table 2 below:

TABLE 2 DRUG NAME CAS NUMBER Acamprosate 77337-76-9; 77337-73-6;107-35-7; 3687-18-1 Aminocaproic Acid 60-32-2 Baclofen 1134-47-0;66514-99-6; 69308-37-8; 70206-22-3; 63701-56-4; 63701-55-3; 847353-30-4Cabergoline 81409-90-7 Carbenoxolone 5697-56-3 or 7421-40-1 Cinnarizine298-57-7 Diethylcarbamazine 90-89-1 or 1642-54-2 Dyphylline 479-18-5Eplerenone 107724-20-9 Etomidate 33125-97-2 Fenoldopam 67227-57-0 or67227-56-9 Leflunomide 75706-12-6 Levosimendan 141505-33-1 Methimazole60-56-0 Moxifloxacin 151096-09-2 or 186826-86-8 or 192927- 63-2 or354812-41-2 Phenformin 114-86-3 or 834-28-6 Prilocaine 721-50-6 or14289-31-7 or 14289-32-8 Quinacrine 83-89-6 or 69-05-6 or 6151-30-0Sulodexide 57821-29-1 Terbinafine 91161-71-6 Trimetazidine 5011-34-7 or13171-25-0 Zonisamide 68291-97-4

Specific examples of prodrugs of Baclofen are given in Hanafi et al,2011 (41), particularly Baclofen esters and Baclofen ester carbamateswhich are of particular interest for CNS targeting. Hence such prodrugsare particularly suitable for compositions of this invention. Arbaclofenplacarbil as mentioned before is also a well-known prodrug and may thusbe used instead of Baclofen in compositions of the invention. Otherprodrugs of Baclofen can be found in the following patent applications:WO2010102071, US2009197958, WO2009096985, WO2009061934, WO2008086492,US2009216037, WO2005066122, US2011021571, WO2003077902, andWO2010120370.

Useful prodrugs for acamprosate such as pantoic acid ester neopentylsulfonyl esters, neopentyl sulfonyl esters prodrugs or maskedcarboxylate neopentyl sulfonyl ester prodrugs of acamprosate are notablylisted in WO2009033069, WO2009033061, WO2009033054 WO2009052191,WO2009033079, US 2009/0099253, US 2009/0069419, US 2009/0082464, US2009/0082440, and US 2009/0076147.

In a preferred embodiment, the invention relates to a compositioncomprising:

at least one first compound selected from Torasemide, Trimetazidine,Mexiletine, Ifenprodil, Bromocriptine and Moxifloxacin salt(s),prodrug(s), derivative(s) of any chemical purity, or sustained releaseformulation(s) thereof, in combination with

at least one second compound, distinct from said first compound,selected from Sulfisoxazole, Methimazole, Prilocaine, Dyphylline,Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen,Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone,Fenoldopam, Leflunomide, Levosimendan, Sulodexide, Terbinafine,Zonisamide, Etomidate, Phenformin, Trimetazidine, Mexiletine,Bromocriptine, Ifenprodil, Torasemide and Moxifloxacin, salt(s),prodrug(s), derivative(s) of any chemical purity, or sustained releaseformulation(s) thereof, for use in the treatment of a neurologicaldisorder in a subject in need thereof.

In a particular embodiment, the invention relates to the use of thesedrugs or compositions for treating AD or a related disorder in a subjectin need thereof.

In a particular embodiment, the invention relates to the use of thesedrugs or compositions for treating MS, PD, ALS, HD, neuropathies (forinstance neuropathic pain or alcoholic neuropathy), alcoholism oralcohol withdrawal, or spinal cord injury, in a subject in need thereof.

As disclosed in the examples, composition therapies using one or more ofthe above-listed drugs lead to an efficient correction of Alzheimer'sdisease and other neurological diseases. As illustrated in theexperimental section, compositions comprising at least Torasemide,Trimetazidine, Mexiletine, Ifenprodil, Bromocriptine, and Moxifloxacinprovide substantial therapeutic and biological effect to prevent thetoxic effects of amyloid (Aβ) protein or peptide on human cells.Moreover, in vivo, these compositions lead to an improvement ofcognitive symptoms as well as to an inhibition of molecular pathwaystriggered by Aβ intoxication, within which glutamate excitotoxicity.Hence they represent novel and potent methods for treating such disease.

The experimental section further shows that the above mentionedcompositions are also efficient in synergistically protecting in vitroneuronal cells from glutamate toxicity, and ii) in conferring clinicalbenefit in in vivo models for diseases related to glutamateexcitotoxicity.

More preferably, drug compositions of the invention may comprise 1, 2,3, 4 or 5 distinct drugs, even more preferably 2, 3 or 4 distinct drugsfor combinatorial treatment of Alzheimer's disease (AD), AD relateddisorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic painor alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinalcord injury in a subject in need thereof. In a preferred embodiment, thedrugs of the invention are used in combination(s) for combined, separateor sequential administration, in order to provide the most effectiveeffect.

In a particular embodiment, the composition comprises (i) Torasemide and(ii) a compound selected from Bromocriptine, Baclofen, Sulfisoxazole,Eplerenone or Terbinafine, or a salt, prodrug, derivative, or sustainedrelease formulation of said compounds (i) and (ii).

In another particular embodiment, the composition comprises (i)Trimetazidine and (ii) a compound selected from Baclofen, Cinnarizine,Zonisamide, or Moxifloxacin, or a salt, prodrug, derivative, orsustained release formulation of said compounds (i) and (ii).

According to a further particular embodiment, the composition comprises(i) Moxifloxacin and (ii) a compound selected from Baclofen, Cinacalcet,Zonisamide, Sulfisoxazole, or Trimetazidine, or a salt, prodrug,derivative, or sustained release formulation of said compounds (i) and(ii).

In another further particular embodiment, the composition comprises (i)Mexiletine and (ii) a compound selected from Baclofen, Cinacalcet,Ifenprodil, or levosimendan or a salt, prodrug, derivative, or sustainedrelease formulation of said compounds (i) and (ii).

A particular embodiment also relates to a composition comprising (i)Ifenprodil and (ii) a compound selected from Acamprosate, Levosimendan,or Mexiletine or a salt, prodrug, derivative, or sustained releaseformulation of said compounds (i) and (ii).

Preferred compositions of the invention, for use in the treatment of aneurological disorder such as Alzheimer's disease (AD), AD relateddisorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic painor alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinalcord injury, comprise one of the following drug combinations, forcombined, separate or sequential administration:

Baclofen and Torasemide,

Eplerenone and Torasemide,

Acamprosate and Ifenprodil,

Baclofen and Mexiletine,

Baclofen and Trimetazidine,

Bromocriptine and Sulfisoxazole,

Cinacalcet and Mexiletine,

Cinnarizine and Trimetazidine,

Sulfisoxazole and Torasemide,

Trimetazidine and Zonisamide,

Levosimendan and Mexiletine,

Levosimendan and Ifenprodil,

Levosimendan and Trimetazidine,

Levosimendan and Moxifloxacin,

Terbinafine and Torasemide,

Moxifloxacin and Trimetazidine,

Moxifloxacin and Baclofen,

Moxifloxacin and Cinacalcet,

Moxifloxacin and Zonisamide,

Moxifloxacin and Sulfisoxazole, or

Mexiletine and Ifenprodil.

Examples of preferred compositions according to the invention comprisinga combination of at least three compounds, for combined, separate orsequential administration, are provided below:

Baclofen and Trimetazidine and Torasemide,

Baclofen and Cinacalcet and Torasemide,

Baclofen and Acamprosate and Torasemide,

Levosimendan and Baclofen and Trimetazidine,

Levosimendan and Aminocaproic acid and Trimetazidine,

Levosimendan and Terbinafine and Trimetazidine, or

Levosimendan and Sulfisoxazole and Trimetazidine.

Examples of preferred compositions according to the invention comprisinga combination of at least four compounds, for combined, separate orsequential administration, are provided below:

Sulfisoxazole and Trimetazidine and Torasemide and Zonisamide,

Sulfisoxazole and Mexiletine and Torasemide and Cinacalcet,

Baclofen and Acamprosate and Torasemide and Diethylcarbamazine, or

Baclofen and Acamprosate and Torasemide and Ifenprodil.

As disclosed in the experimental section the above combination therapiesof the invention induce a strong neuroprotective effect against Aβtoxicity and give positive results in behavioural performances andbiochemical assays in vivo. The results show that compositions of theinvention i) efficiently correct molecular pathways triggered, in vivo,by Aβ aggregates and ii) lead to an improvement of neurophysiologicalimpairments observed in diseased animals as neuron survival or synapseintegrity.

Moreover, the results presented show also that the above combinationstherapies have an important synergistic neuroprotecting effect againstglutamate excitotoxicity (FIGS. 24 and 25, table 8), a pathway which isimplicated in various neurological diseases as AD, MS, PD, ALS, HD,neuropathies (for instance neuropathic pain or alcoholic neuropathy),alcoholism or alcohol withdrawal, or spinal cord injury. These therapiesgive positive results in in vivo or in vitro models for these diseases.

In addition, in vivo results also show that compositions of theinvention efficiently restore Brain Blood Barrier integrity, which isknown to be impaired in several neurological diseases.

An object of this invention thus also resides in a composition asdefined above for treating a neurological disorder such as Alzheimer'sdisease (AD), AD related disorders, MS, PD, ALS, HD, neuropathies (forinstance alcoholic neuropathy or neuropathic pain), alcoholism oralcohol withdrawal, or spinal cord injury.

A further object of this invention resides in the use of a compositionas defined above for the manufacture of a medicament for treating aneurological disorder such as Alzheimer's disease (AD), AD relateddisorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic painor alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinalcord injury.

The invention further provides a method for treating a neurologicaldisorder such as Alzheimer's disease (AD), AD related disorders, MS, PD,ALS, HD, neuropathies (for instance neuropathic pain or alcoholicneuropathy), alcoholism or alcohol withdrawal, or spinal cord injury,comprising administering to a subject in need thereof an effectiveamount of a composition as disclosed above.

As indicated previously, the compounds in a combinatorial treatment orcomposition of the present invention may be formulated together orseparately, and administered together, separately or sequentially and/orrepeatedly.

In this regard, a particular object of this invention is a method fortreating AD, an AD related disorder, MS, PD, ALS, HD, neuropathies (forinstance neuropathic pain or alcoholic neuropathy), alcoholism oralcohol withdrawal, or spinal cord injury in a subject, comprisingadministering simultaneously, separately or sequentially to a subject inneed of such a treatment, an effective amount of a composition asdisclosed above.

In a preferred embodiment, the invention relates to a method of treatingAlzheimer's disease (AD), an AD related disorder, MS, PD, ALS, HD,neuropathies (for instance neuropathic pain or alcoholic neuropathy),alcoholism or alcohol withdrawal, or spinal cord injury in a subject inneed thereof, comprising administering to the subject an effectiveamount of Torasemide, Trimetazidine, Mexiletine, Ifenprodil,Bromocriptine or Moxifloxacin, or salt(s) or prodrug(s) or derivative(s)or sustained release formulation(s) thereof, preferably in a combinationas disclosed above.

In another embodiment, this invention relates to a method of treatingAlzheimer's disease (AD), an AD related disorder, MS, PD, ALS, HD,neuropathies (for instance neuropathic pain or alcoholic neuropathy),alcoholism or alcohol withdrawal, or spinal cord injury in a subject inneed thereof, comprising simultaneously, separately or sequentiallyadministering to the subject at least one first compound selected fromthe group consisting of Torasemide, Trimetazidine, Mexiletine,Ifenprodil, Bromocriptine and Moxifloxacin salts, prodrugs, derivatives,or any formulation thereof, in combination with at least one secondcompound distinct from said first compound, selected from,Sulfisoxazole, Methimazole, Prilocaine, Dyphylline, Quinacrine,Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen, Cabergoline,Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam,Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide,Etomidate, Phenformin, Trimetazidine, Mexiletine, Bromocriptine,Ifenprodil, Torasemide, and Moxifloxacin salts, prodrugs, derivatives,or any formulation thereof.

In a further embodiment, the invention relates to a method of treatingAlzheimer's disease (AD), an AD related disorder, MS, PD, ALS, HD,neuropathies (for instance neuropathic pain or alcoholic neuropathy),alcoholism or alcohol withdrawal, or spinal cord injury comprisingadministering to a subject in need thereof, at least one first compoundselected from the group consisting of Torasemide, Trimetazidine,Mexiletine, Ifenprodil, Bromocriptine and Moxifloxacin salts, prodrugs,derivatives, or any formulation thereof, in combination with at leastone second compound distinct from said first compound, selected from,Sulfisoxazole, Methimazole, Prilocaine, Dyphylline, Quinacrine,Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen, Cabergoline,Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam,Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide,Etomidate, Phenformin, Trimetazidine, Mexiletine, Bromocriptine,Ifenprodil, Torasemide, and Moxifloxacin salts, prodrugs, derivatives,or any formulation thereof.

As disclosed in the examples, besides being efficient in protectingneurons from glutamate toxicity, baclofen-torasemide therapy is alsoparticularly efficient in promoting neuronal cell growth, even in theabsence of any exposure to a toxic agent or condition. Moreover, invivo, this combination therapy leads to an improvement of loss oftransduction of nervous signal subsequent to nerve injury. Hencebaclofen-torasemide combination represents a novel and potent therapyfor treating neuropathies, nerve injuries, and spinal cord injury asdefined above.

In this regard, in a particular embodiment, the invention relates to amethod for treating neuropathies, e.g., peripheral nerves injuries orspinal cord injuries, comprising administering to a subject in needthereof a composition comprising baclofen and torasemide salts,prodrugs, derivatives, or any formulation thereof.

In another particular embodiment, the invention relates to a method fortreating neuropathies, e.g., inherited neuropathies as CMT diseases,comprising administering to a subject in need thereof a compositioncomprising baclofen and torasemide, or salts, prodrugs, derivatives, orany formulation thereof.

In a more particular embodiment, the invention relates to a method fortreating CMT1 or CMT2 disease, comprising administering to a subject inneed thereof a composition comprising baclofen and torasemide, or salts,prodrugs, derivatives, or any formulation thereof.

A particular object of this invention is also a method for treatingneuropathies, e.g., peripheral nerves injuries or spinal cord injuries,comprising administering simultaneously, separately or sequentiallyand/or repeatedly to a subject in need of such a treatment, an effectiveamount of a baclofen and torasemide as disclosed above.

Although very effective in vitro and in vivo, depending on the subjector specific condition, the methods and compositions of the invention maybe used in further conjunction with additional drugs or treatmentsbeneficial to the treated neurological condition in the subjects. Inthis regard, in a particular embodiment, the drug(s) or compositionsaccording to the present invention may be further combined with Ginkgobiloba extracts. Suitable extracts include, without limitation, Ginkgobiloba extracts, improved Ginkgo biloba extracts (for example enrichedin active ingredients or lessened in contaminant) or any drug containingGinkgo biloba extracts.

Ginkgo biloba extracts may be used in a composition comprising at leastTorasemide, Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil andMoxifloxacin.

In preferred embodiments, Ginkgo Biloba extracts are used in combinationwith anyone of the following drug combinations:

Acamprosate and Ifenprodil,

Baclofen and Mexiletine,

Baclofen and Torasemide,

Baclofen and Trimetazidine,

Bromocriptine and Sulfisoxazole,

Cinacalcet and Mexiletine,

Cinnarizine and Trimetazidine,

Eplerenone and Torasemide,

Sulfisoxazole and Torasemide,

Trimetazidine and Zonisamide,

Levosimendan and Mexiletine,

Levosimendan and Ifenprodil,

Levosimendan and Trimetazidine,

Levosimendan and Moxifloxacin,

Terbinafine and Torasemide,

Moxifloxacin and Baclofen,

Moxifloxacin and Cinacalcet,

Moxifloxacin and Zonisamide,

Moxifloxacin and Sulfisoxazole,

Mexiletine and Ifenprodil,

Baclofen and Trimetazidine and Torasemide,

Baclofen and Cinacalcet and Torasemide,

Baclofen and Acamprosate and Torasemide,

Sulfisoxazole and Trimetazidine and Torasemide and Zonisamide,

Sulfisoxazole and Mexiletine and Torasemide and Cinacalcet,

Baclofen and Acamprosate and Torasemide and Diethylcarbamazine,

Baclofen and Acamprosate and Torasemide and Ifenprodil,

Levosimendan and Baclofen and Trimetazidine,

Levosimendan and Aminocaproic acid and Trimetazidine,

Levosimendan and Terbinafine and Trimetazidine, or

Levosimendan and Sulfisoxazole and Trimetazidine.

Other therapies used in conjunction with drug(s) or drug(s)combination(s) according to the present invention, may comprise one ormore drug(s) that ameliorate symptoms of Alzheimer's disease, an ADrelated disorder, MS, PD, ALS, HD, neuropathies (for instanceneuropathic pain or alcoholic neuropathy), alcoholism or alcoholwithdrawal, or spinal cord injury, or drug(s) that could be used forpalliative treatment of these disorders.

For instance, combinations of the invention can be used in conjunctionwith Donepezil (CAS: 120014-06-4), Gabapentine (CAS: 478296-72-9;60112-96-3), Galantamine (357-70-0), Rivastigmine (123441-03-2) orMemantine (CAS: 19982-08-2).

A further object of this invention relates to the use of a compound orcombination of compounds as disclosed above for the manufacture of amedicament for the treatment of the above listed disorders, by combined,separate or sequential administration to a subject in need thereof.

A further object of this invention is a method of preparing apharmaceutical composition, the method comprising mixing the abovecompounds in an appropriate excipient or carrier.

The duration of the therapy depends on the stage of the disease ordisorder being treated, the combination used, the age and condition ofthe patient, and how the patient responds to the treatment. The dosage,frequency and mode of administration of each component of thecombination can be controlled independently. For example, one drug maybe administered orally while the second drug may be administeredintramuscularly. Combination therapy may be given in on-and-off cyclesthat include rest periods so that the patient's body has a chance torecover from any as yet unforeseen side-effects. The drugs may also beformulated together such that one administration delivers all drugs.

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to ameliorate the patientcondition or efficiently treat the disease or disorder.

While it is possible for the active ingredients of the combination to beadministered as the pure chemical it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulations. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp using the formulation most suitably for the treatment. Suchmeasures make a patient pack specifically suitable for and adapted foruse for treatment with the combination of the present invention.

The drug may be contained, in any appropriate amount, in any suitablecarrier substance (e.g., excipient, vehicle, support), which mayrepresent 1-99% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable for theoral, parenteral (e.g., intravenously, intramuscularly), rectal,cutaneous, nasal, vaginal, inhalant, skin (patch), or ocularadministration route. Thus, the composition may be in the form of, e.g.,tablets, capsules, pills, powders, granulates, suspensions, emulsions,solutions, gels including hydrogels, pastes, ointments, creams,plasters, drenches, osmotic delivery devices, suppositories, enemas,injectables, implants, sprays, or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overau extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time; (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively, constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug, either alone or incombination, has (i) a narrow therapeutic index (i.e., the differencebetween the plasma concentration leading to harmful side effects ortoxic reactions and the plasma concentration leading to a therapeuticeffect is small; in general, the therapeutic index, TI, is defined asthe ratio of median lethal dose (LD50) to median effective dose (ED50));(ii) a narrow absorption window in the gastro-intestinal tract; or (iii)a very short biological half-life so that frequent dosing during a dayis required in order to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, various types of controlled release compositionsand coatings. Thus, the drug is formulated with appropriate excipientsinto a pharmaceutical composition that, upon administration, releasesthe drug in a controlled manner (single or multiple unit tablet orcapsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, microcrystalline cellulose, starches includingpotato starch, calcium carbonate, sodium chloride, calcium phosphate,calcium sulfate, or sodium phosphate); granulating and disintegratingagents (e.g., cellulose derivatives including microcrystallinecellulose, starches including potato starch, croscarmellose sodium,alginates, or alginic acid); binding agents (e.g., acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., stearic acid, silicas, or talc).Other pharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methyl call lose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology.

Several drugs may be mixed together in the tablet, or may bepartitioned. For example, the first drug is contained on the inside ofthe tablet, and the second drug is on the outside, such that asubstantial portion of the second drug is released prior to the releaseof the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin, or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally byinjection, infusion or implantation (intravenous, intramuscular,subcutaneous, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation or such compositions are well known to those skilled in theart of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in theform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polyglactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine). Biocompatible carriersthat may be used when formulating a controlled release parenteralformulation are carbohydrates (e.g., dextrans), proteins (e.g.,albumin), lipoproteins, or antibodies. Materials for use in implants canbe non-biodegradable (e.g., polydimethyl siloxane) or biodegradable(e.g., poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).

Alternative Routes

Although less preferred and less convenient, other administrationroutes, and therefore other formulations, may be contemplated. In thisregard, for rectal application, suitable dosage forms for a compositioninclude suppositories (emulsion or suspension type), and rectal gelatincapsules (solutions or suspensions). In a typical suppositoryformulation, the active drug(s) are combined with an appropriatepharmaceutically, acceptable suppository base such as cocoa butter,esterified fatty acids, glycerinated gelatin, and various water-solubleor dispersible bases like polyethylene glycols. Various additives,enhancers, or surfactants may be incorporated.

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and Liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The preservatives, humectants, penetration enhancers may be parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride,glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor alternatively plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. If there is sequentialadministration, the delay in administering the second (or additional)active ingredient should not be such as to lose the benefit of theefficacious effect of the combination of the active ingredients. Aminimum requirement for a combination according to this description isthat the combination should be intended for combined use with thebenefit of the efficacious effect of the combination of the activeingredients. The intended use of a combination can be inferred byfacilities, provisions, adaptations and/or other means to help using thecombination according to the invention.

Although the active drugs of the present invention may be administeredin divided doses, for example two or three times daily, a single dailydose of each drug in the combination is preferred, with a single dailydose of all drugs in a single pharmaceutical composition (unit dosageform) being most preferred.

The term “unit dosage form” refers to physically discrete units (such ascapsules, tablets, or loaded syringe cylinders) suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material or materials calculated to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier.

Administration is generally repeated. It can be one to several timesdaily for several days to several years, and may even be for the life ofthe patient. Chronic or at least periodically repeated long-termadministration is indicated in most cases.

Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular patient may affect the dosage used.

Except when responding to especially impairing cases when higher dosagesmay be required, the preferred dosage of each drug in the combinationusually lies within the range of doses not above those usuallyprescribed for long-term maintenance treatment or proven to be safe inphase 3 clinical studies.

One remarkable advantage of the invention is that each compound may beused at low doses in a combination therapy, while producing, incombination, a substantial clinical benefit to the patient. Thecombination therapy may indeed be effective at doses where the compoundshave individually no substantial effect. Accordingly, a particularadvantage of the invention lies in the ability to use sub-optimal dosesof each compound, i.e., doses which are lower than therapeutic dosesusually prescribed, preferably ½ of therapeutic doses, more preferably⅓, ¼, ⅕, or even more preferably 1/10 of therapeutic doses. Inparticular examples, doses as low as 1/20, 1/30, 1/50, 1/100, or evenlower, of therapeutic doses are used.

At such sub-optimal dosages, the compounds alone would be substantiallyinactive, while the combination(s) according to the invention are fullyeffective.

A preferred dosage corresponds to amounts from 1%, up to 50% of thoseusually prescribed for long-term maintenance treatment.

The most preferred dosage may correspond to amounts from 1% up to 10% ofthose usually prescribed for long-term maintenance treatment.

Specific examples of dosages of drugs for use in the invention areprovided below:

-   -   Bromocriptine orally from about 0.01 to 10 mg per day,        preferably less than 5 mg per day, more preferably less than 2.5        mg per day, even more preferably less than 1 mg per day, such        dosages being particularly suitable for oral administration,    -   Ifenprodil orally from about 0.4 to 6 mg per day, preferably        less than 3 mg per 2.5 day, more preferably less than 1.5 mg per        day, even more preferably less than 0.75 mg per day, such        dosages being particularly suitable for oral administration,    -   Mexiletine orally from about 6 to 120 mg per day, preferably        less than 60 mg per day, more preferably less than 30 mg per        day, even more preferably less than 15 mg per day, such dosages        being particularly suitable for oral administration,    -   Moxifloxacin orally from about 4 to 40 mg per day, preferably        less than 20 mg per day, more preferably less than 10 mg per        day, even more preferably less than 5 mg per day, such dosages        being particularly suitable for oral administration,    -   Torasemide orally from about 0.05 to 4 mg per day, preferably        less than 2 mg per day, more preferably less than 1 mg per day,        even more preferably less than 0.5 mg per day, such dosages        being particularly suitable for oral administration,    -   Trimetazidine orally from about 0.4 to (5 mg per day, preferably        less than 3 mg per day, more preferably less than 1.5 mg per        day, even more preferably less than 0.75 mg per day, such        dosages being particularly suitable for oral administration,    -   Acamprosate orally from about 1 to 400 mg per day,    -   Aminocaproic Acid orally from about 0.1 g to 2.4 g per day,    -   Baclofen orally from about 0.15 to 15 mg per day,    -   Diethylcarbamazine orally from about 0.6 to 600 mg per day,    -   Cinacalcet orally from about 0.3 to 36 mg per day,    -   Cinnarizine orally from about 0.6 to 23 mg per day,    -   Eplerenone orally from about 0.25 to 10 mg per day,    -   Leflunomide orally from about 0.1 to 10 mg per day,    -   Levosimendan orally from about 0.04 to 0.8 mg per day,    -   Sulfisoxazole orally from about 20 to 800 mg per day,    -   Sulodexide orally from about 0.05 to 40 mg per day,    -   Terbinafine orally from about 2.5 to 25 mg per day,    -   Zonisamide orally from about 0.5 to 50 mg per day.

It will be understood that the amount of the drug actually administeredwill be determined by a physician, in the light of the relevantcircumstances including the condition or conditions to be treated, theexact composition to be administered, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thechosen route of administration. Therefore, the above dosage ranges areintended to provide general guidance and support for the teachingsherein, but are not intended to limit the scope of the invention.

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLES

The Care and Husbandry of Animals as Well as the Experimentations arePerformed According to the Guidelines of the Committee Far Research andEthical Issue of the I.A.S.P. (1983).

A) Treatment of Diseases Related to Aβ Toxicity

In this series of experiments, candidate compounds have been tested fortheir ability to prevent or reduce the toxic effects of human Aβ₁₋₄₂.Aβ₁₋₄₂ is the fill length peptide that constitutes aggregates found inbiopsies from human patients afflicted with AD, The drugs are firsttested individually, followed by assays of their combinatorial action.The effect is determined on various cell types, to further document theactivity of the compounds in in vitro models which illustrate differentphysiological features of AD. In vivo studies are also performed in amouse model for AD confirming this protective effect by evaluating theeffect of the compounds on i) the cognitive performance of animals andii) on molecular hallmarks (apoptosis induction, oxidative stressinduction, inflammation pathway induction) of AD.

I. The Compounds Prevent Toxicity of Human Aβ₁₋₄₂ I.1. ProtectionAgainst the Toxicity of Aβ₁₋₄₂ in Human Brain Microvascular EndothelialCell Model

Human brain microvascular endothelial cell cultures were used to studythe protection afforded by candidate compound(s) on Aβ₁₋₄₂ toxicity.

Human brain microvascular endothelial cerebral cells (HBMEC, ScienCellRef: 1000, frozen at passage 10) were rapidly thawed in a waterbath at±37° C. The supernatant was immediately put in 9 ml Dulbecco's modifiedEagle's medium (DMEM; Pan Biotech ref: P04-03600) containing 10% offoetal calf serum (FCS; GIBCO ref 10270-106). Cell suspension wascentrifuged at 180×g for 10 min. at +4° C. and the pellets weresuspended in CSC serum-free medium (CSC serum free, Cell System, Ref:SF-4Z0-500-R, Batch 51407-4) with 1.6% of Serum free RocketFuel (CellSystem, Ref: SF-4Z0-500-R, Batch 54102), 2% of Penicillin 10.000 U/mland Streptomycin 10 mg/ml (PS; Pan Biotech ref P06-07100 batch133080808) and were seeded at the density of 20000 cells per well in 96well-plates (matrigel layer biocoat angiogenesis system, BD, Ref 354150,Batch A8662) in a final volume of 100 μl. On matrigel support,endothelial cerebral cells spontaneously started the process ofcapillary network morphogenesis (33).

Three separate cultures were performed per condition, 6 wells percondition.

Candidate Compounds and Human Amyloid β₁₋₄₂ Treatment

Briefly, Aβ₁₋₄₂ peptide (Bachem, ref: H1368 batch 1010533) wasreconstituted in define culture medium at 20 μM (mother solution) andwas slowly shaken at +37° C. for 3 days in dark for aggregation. Thecontrol medium was prepared in the same conditions.

After 3 days, this aggregated human amyloid peptide was used on HBMEC at2.5 μM diluted in control medium (optimal incubation time). The Aβ₁₋₄₂peptide was added 2 hours after HBMEC seeding on matrigel for 18 hoursincubation.

One hour after HBMEC seeding on matrigel, test compounds and VEGF-165were solved in culture medium (+0.1% DMSO) and then pre-incubated withHBMEC for 1 hour before the Aβ₁₋₄₂ application (in a final volume perculture well of 1000). One hour after test compounds or VEGF incubation(two hours after cell seeding on matrigel), 100 μl of Aβ₁₋₄₂ peptide wasadded to a final concentration of 2.50 μM diluted in control medium inpresence of test compounds or VEGF (in a 200 μl total volume/well), inorder to avoid further drug dilutions.

Organization of Cultures Plates

VEGF-165 known to be a pro-angiogenic isoform of VEGF-A, was used forall experiment in this study as reference compound. VEGF-165 is one ofthe most abundant VEGF isoforms involved in angiogenesis. VEGF was usedas reference test compound at 10 nM.

The following conditions were assessed:

-   -   Negative Control: medium alone+0.1% DMSO    -   Intoxication: amyloid-β₁₋₄₂ (2.5 μM) for 18 h    -   Positive control: VEGF-165 (10 nM) (1 reference        compound/culture) 1 hr before the Aβ₁₋₄₂ (2.5 μM) addition for a        18 h incubation time.    -   Test compounds. Test compound 1 hr before the Aβ₁₋₄₂ (2.5 μM)        addition for a 18 h incubation time.

Capillary Network Quantification

Per well, 2 pictures with 4× lens were taken using IInCell Analyzer™1000 (GE Healthcare) in light transmission. All images were taken in thesame conditions. Analysis of the angiogenesis networks was done usingDeveloper software (GE Healthcare). The total length of capillarynetwork was assessed.

Data Processing

All values are expressed as mean±s.e. mean of the 3 cultures (n=6 percondition). Statistical analyses were done on the different conditionsperforming an ANOVA followed by the Dunnett's test when it was allowed(Statview software version 5.0). The values (as %) inserted on thegraphs show the amyloid toxicity evolution. Indeed, the amyloid toxicitywas taken as the 100% and the test compound effect was calculated as a %of this amyloid toxicity.

Results

Results are shown in FIG. 1. They demonstrate that the drugs testedalone, induce a substantial protective effect against the toxicitycaused by Aβ peptide 1-42:

-   -   Torasemide, at a low dosage of e.g., 400 nM, induces strong        protective effect;    -   Bromocriptine, at a low dosage of e.g., 3.2 nM, induces strong        protective effect.

The results also show that, unexpectedly, upper or lower drugconcentrations in comparison to the above mentioned drug concentrations,may worsen or rather have less to no effect on Aβ₁₋₄₂ toxicity in thismodel.

I.2 Protection Against the Toxicity of Aβ₁₋₄₂ on Primary Cortical NeuronCells. Test Compound and Human Amyloid-β1-42 Treatment

Rat cortical neurons were cultured as described by Singer et al. (42).Briefly pregnant female rats of 15 days gestation were killed bycervical dislocation (Rats Wistar) and the fetuses were removed from theuterus. The cortex was removed and placed in ice-cold medium ofLeibovitz (L15) containing 2% of Penicillin 10.000 U/ml and Streptomycin1.0 mg/ml and 1% of bovine serum albumin (BSA). Cortices weredissociated by trypsin for 20 min at 37° C. (0.05%), The reaction wasstopped by the addition of Dulbecco's modified Eagle's medium (DMEM)containing DNase1 grade II and 10% of foetal calf serum (FCS). Cellswere then mechanically dissociated by 3 serial passages through a 10 mlpipette and centrifuged at 515×g for 10 min at +4° C., The supernatantwas discarded and the pellet of cells was re-suspended in a definedculture medium consisting of Neurobasal supplemented with B27 (2%),L-glutamine (0.2 mM), 2% of PS solution and 10 ng/ml of BDNF. Viablecells were counted in a Neubauer cytometer using the trypan blueexclusion test. The cells were seeded at a density of 30000 cells/wellin 96 well-plates (wells were pre-coated with poly-L-lysine (10 μg/ml))and were cultured at ±37° C. in a humidified air (95%)/CO2 (5%)atmosphere.

Briefly, Aβ₁₋₄₂ peptide was reconstituted in define culture medium at 40μM (mother solution) and was slowly shook at 1-37° C. for 3 days in darkfor aggregation. The control medium was prepared in the same conditions.

After 3 days, the solution was used on primary cortical neurons asfollows:

After 10 days of neuron culture, drug was solved in culture medium(+0.1% DMSO) and then pre-incubated with neurons for 1 hour before theAβ₁₋₄₂ application (in a final volume per culture well of 100 μl). Onehour after drug(s) incubation, 100 μl of Aβ₁₋₄₂ peptide was added to afinal concentration of 10 μM diluted in presence of drug(s), in order toavoid further drug(s) dilutions. Cortical neurons were intoxicated for24 hours. Three separate cultures were performed per condition, 6 wellsper condition. BDNF (50 ng/ml) and Estradiol-β (150 nM) were used aspositive control and reference compounds respectively.

Organization of Cultures Plates

Estradiol-β at 150 nM was used as a positive control.

Estradiol-β was solved in culture medium and pre-incubated for 1 hbefore t aggregated amyloid-β₁₋₄₂ application.

The following conditions were assessed:

CONTROL PLAQUE: 12 wells/condition

-   -   Negative Control: medium alone+0.1% DMSO    -   intoxication: amyloid-β₁₋₄₂ (10 UM) for 24 h    -   Reference compound: Estradiol (150 nM) 1 hr.

DRUG PLATE: 6 wells/condition

-   -   Negative Control: medium alone+0.1% DMSO    -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h    -   Drug: Drug—1 hr followed by amyloid-β₁₋₄₂ (10 μM) for 24 h

Lactate Dehydrogenase (LDH) Activity Assay

24 hours after intoxication, the supernatant was taken off and analyzedwith Cytotoxicity Detection Kit (LDH, Roche Applied Science, ref11644793001, batch: 11800300). This colorimetric assay for thequantification of cell toxicity is based on the measurement of lactatedehydrogenase (LDH) activity released from the cytosol of dying cellsinto the supernatant.

Data Processing

All values are expressed as mean±s.e. mean of the 3 cultures (n=6 percondition). Statistic analyses were done on the different conditions(ANOVA followed by the Dunnett's test when it was allowed, Statviewsoftware version 5.0).

Results

The results obtained for individual selected drugs in the toxicityassays on primary cortical neuron cells are presented in FIGS. 2, 26 and27. They demonstrate that the drugs tested alone, induce a substantialprotective effect against the toxicity caused by Aβ peptide 1-42:

-   -   Trimetazidine, at a low dosage of e.g., 40 nM, induces strong        protective effect;    -   Mexiletine, at a dose as low as 3.2 nM, induces a strong        protective effect;    -   Bromocriptine, at a dose as low as 40 nM, induces a strong        protective effect;    -   Ifenprodil, at a dose as low as 600 nM, induces a strong        protective effect;    -   Moxifloxacin, at a dose as low as 20 nM, induces a strong        protective effect.    -   Torasemide, at a dose of 200 nM, induces a strong protective        effect.    -   Homotaurine, at a dose of 8 nM, induces a strong protective        effect.

The obtained results also show that, unexpectedly, upper or lower drugconcentrations than those indicated above, may worsen or rather haveless to no protective effect on Aβ₁₋₄₂ toxicity for neuronal cells.

II. Combined Therapies Prevent Toxicity of Human Aβ₁₋₄₂ II.1 Effect ofCombined Therapies on the Toxicity of Human Aβ₁₋₄₂ Peptide on HumanHBMEC Cells.

The efficacy of drug combinations of the invention is assessed on humancells. The protocol which is used in these assays is the same asdescribed in section I.1 above.

Results

All of the tested drug combinations give protective effect againsttoxicity of human Aβ₁₋₄₂ peptide in HBMEC model, as shown in table 3below and exemplified in FIGS. 3 to 6 and FIGS. 13 and 14. The resultsclearly show that the intoxication by, aggregated human amyloid peptide(Aβ₁₋₄₂ 2.5 μm) is significantly prevented by combinations of theinvention whereas, at those concentrations, drugs alone have nosignificant effect on intoxication in the experimental conditionsdescribed above.

TABLE 3 Protective effect in Aβ₁₋₄₂ intoxicated DRUG COMBINATION HBMECcells Baclofen and Torasemide + Eplerenone and Torasemide +Bromocriptine and Sulfisoxazole + Sulfisoxazole and Torasemide +Terbinafine and Torasemide + Mexiletine and Cinacalcet + Baclofen andTrimetazidine and Torasemide + Baclofen and Cinacalcet and Torasemide +Baclofen and Acamprosate and Torasemide + Sulfisoxazole andTrimetazidine and Torasemide + and Zonisamide Sulfisoxazole andMexiletine and Torasemide and + Cinacalcet Baclofen and Acamprosate andTorasemide and + Diethylcarbamazine Baclofen and Acamprosate andTorasemide + and Ifenprodil Levosimendan and Baclofen andTrimetazidine + Levosimendan and Aminocaproic acid and + TrimetazidineLevosimendan and Terbinafine and Trimetazidine + Levosimendan andSulfisoxazole and Trimetazidine +

As exemplified in FIGS. 3 to 6, 13 and 14, the following drugcombinations give particularly interesting protective effects againsttoxicity of human Aβ₁₋₄₂ peptide in intoxicated HBMEC cells:

-   -   Baclofen and Torasemide,    -   Sulfisoxazole and Torasemide,    -   Torasemide and Eplerenone,    -   Sulfisoxazole and Bromocriptine.    -   Terbinafine and Torasemide, or    -   Cinacalcet and Mexiletine.

II.2 Effect of Combined Therapies on the Toxicity of Human Aβ₁₋₄₂Peptide on Primary Cortical Neuron Cells.

The efficacy of drug combinations of the invention is assessed onprimary cortical neuron cells. The protocol which is used in theseassays is the same as described in section I.2 above.

Results

All of the tested drug combinations give protective effect againsttoxicity of human Aβ₁₋₄₂ peptide in primary cortical neuron cells, asshown in Table 4 below and exemplified in FIGS. 7 to 12 and 16 to 22.The results clearly show that the intoxication by aggregated humanamyloid peptide (Aβ₁₋₄₂ 10 μM) is significantly prevented bycombinations of the invention whereas, at those concentrations, drugsalone have no significant effect on intoxication in the experimentalconditions described above.

TABLE 4 Protective effect in Aβ₁₋₄₂ intoxicated primary cortical DRUGCOMBINATIONS neuron cells Acamprosate and Ifenprodil + Baclofen andMexiletine + Baclofen and Trimetazidine + Baclofen and Torasemide +Cinacalcet and Mexiletine + Cinnarizine and Trimetazidine +Trimetazidine and Zonisamide + Levosimendan and Mexiletine +Levosimendan and Ifenprodil + Levosimendan and Trimetazidine +Levosimendan and Moxifloxacin + Mexiletine and Ifenprodil + Moxifloxacinand Baclofen + Moxifloxacin and Cinacalcet + Moxifloxacin andTrimetazidine + Moxifloxacin and Sulfisoxazole + Moxifloxacin andZonisamide + Torasemide and Sulfisoxazole + Baclofen and Trimetazidineand Torasemide + Baclofen and Cinacalcet and Torasemide + Baclofen andAcamprosate and Torasemide + Sulfisoxazole and Trimetazidine andTorasemide + and Zonisamide Sulfisoxazole and Mexiletine and Torasemideand + Cinacalcet Baclofen and Acamprosate and Torasemide and +Diethylcarbamazine Baclofen and Acamprosate and Torasemide and +Ifenprodil Levosimendan and Baclofen and Trimetazidine + Levosimendanand Aminocaproic acid and + Trimetazidine Levosimendan and Terbinafineand Trimetazidine + Levosimendan and Sulfisoxazole and + Trimetazidine

As exemplified in FIGS. 7 to 12 and 15 to 22, the following drugcombinations give particularly interesting protective effects againsttoxicity of human Aβ₁₋₄₂ peptide in intoxicated primary cortical neuroncells:

-   -   Acamprosate and Ifenprodil,    -   Baclofen and Mexiletine,    -   Baclofen and Torasemide,    -   Baclofen and Trimetazidine,    -   Cinacalcet and Mexiletine,    -   Cinnarizine and Trimetazidine,    -   Trimetazidine and Zonisamide,    -   Mexiletine and Ifenprodil,    -   Moxifloxacin and Baclofen,    -   Moxifloxacin and Cinacalcet,    -   Moxifloxacin and Trimetazidine,    -   Moxifloxacin and Sulfisoxazole,    -   Moxifloxacin and Zonisamide, or    -   Torasemide and Sulfisoxazole.

II.4. Protection of Neurite Growth Against Aβ₁₋₄₂ Toxicity. TestCompounds and Aβ1-42 Treatment

Primary rat cortical neurons are cultured as described previously.

After 10 days of culture, cells are incubated with drugs. After 1 hour,cells are intoxicated by 2.5 μM of beta-amyloid (1-42; Bachem) indefined medium without BDNF but together with drugs. Cortical neuronsare intoxicated for 24 hours. BDNF (10 ng/ml) is used as a positive(neuroprotective) control. Three independent cultures were performed percondition, 6 wells per condition.

Neurites Length

After 24 hours of intoxication, the supernatant is taken off and thecortical neurons are fixed by a cold solution of ethanol (95%) andacetic acid (5%) for 5 min, After permeabilization with 0.1% of saponin,cells are blocked for 2 h with PBS containing 1% foetal calf serum.Then, cells are incubated with monoclonal antibody antimicrotubule-associated-protein 2 (MAP-2; Sigma). This antibody isrevealed with Alexa Fluor 488 goat anti-mouse IgG (Molecular probe).Nuclei of neurons were labeled by a fluorescent marker (Hoechstsolution, SIGMA).

Per well, 10 pictures are taken using InCell Analyzer™ 1000 (GEHealthcare) with 20× magnification. All pictures are taken in the sameconditions. Analysis of the neurite network is done using Developersoftware (GE Healthcare) in order to assess the total length of neuritenetwork.

Results

The combination of Baclofen and Torasemide induces a significantprotective effect against the toxicity of human Aβ₁₋₄₂ peptide(improvement of 531% of neurites network) in primary cortical neuroncells as shown in FIG. 23. The results clearly show that theintoxication by human amyloid peptide (Aβ₁₋₄₂ 2.5 μM) is significantlyprevented by the combination and that, moreover, the combinationenhances neurite network in comparison with control.

Hence, this combination allows an effective protection of corticalneuron cells and of cell neuronal networks against the toxicity of humanAβ₁₋₄₂ peptide. Moreover, such an augmentation of neurites networkconfirms the efficacy of such drugs in neurological disorders likespinal cord injury.

III. The Compounds Prevent Toxicity of Human Aβ₂₅₋₃₅ In Vivo Animals

Male Swiss mice are used throughout the study. Animals are housed inplastic cages, with free access to laboratory chow and water, exceptduring behavioural experiments, and kept in a regulated environment,under a 12 h light/dark cycle (light on at 8:00 a.m.). Behavioralexperiments are carried out in a soundproof and air-regulatedexperimental room, to which mice have been habituated at least 30 minbefore each experiment.

Amyloid Peptide Preparation and Infection

The Aβ₂₅ ₃₅ peptide and scrambled Aβ₂₅ ₃₅ peptide have been dissolved insterile bidistilled water, and stored at −20° C. until use. Lightmicroscopic observation indicated that incubating the Aβ₂₅₋₃₅ peptide,but not the scrambled Aβ₂₅₋₃₅ peptide, led the presence of two types ofinsoluble precipitates, birefringent fibril-like structures andamorphous globular aggregates. The ii-amyloid peptides are thenadministered intracerebroventricularly (i.c.v.). In brief, each mouse isanaesthetized lightly with ether, and a gauge stainless-steel needle isinserted unilaterally 1 mm to the right of the midline point equidistantfrom each eye, at an equal distance between the eyes and the ears andperpendicular to the plane of the skull. Peptides or vehicle aredelivered gradually within approximately 3 s. Mice exhibit normalbehaviour within 1 min after injection. The administration site ischecked by injecting Indian ink in preliminary experiments. Neitherinsertion of the needle, nor injection of the vehicle have had asignificant influence on survival, behavioral responses or cognitivefunctions.

Drug(s) Treatment

On day −1, i.e. 24 h before the Aβ₂₅₋₃₅ peptide injection, drugs, drugscombination or the vehicle solution are administered per os by gavagetwice daily (at 8:00 am and 6:00 pm).

On day 0 (at 10:00 am), mice are injected i.c.v. with Aβ25-35 peptide orscrambled Aβ 25-35 peptide (control) in a final volume of 3 μl (3 mM).

Between day 0 and day 7, drugs, drugs combination or the vehiclesolution are administered per os by gavage once or twice daily (at 8:00am and 6:00 pm). One animal group receives donepezil (referencecompound—1 mg/kg/day) per os by gavage in a single injection (at 8:00am). Drugs are solubilized in water and freshly prepared just beforeeach gavage administration.

On day 7, all animals are tested for the spontaneous alternationperformance in the Y-maze test, an index of spatial working memory.

On day 7 and 8, the contextual long-term memory of the animals isassessed using the step-down type passive avoidance procedure.

On day 8, animals are sacrificed. Their brain is dissected and kept at−80° C. for further analysis.

Positive results are observed in behavioral performances and biochemicalassays performed 7 days after Aβ₂₅₋₃₅ peptide icy injection, notably forthe combinations listed in table 5.

TABLE 5 Results in biochemical and/or behavioral DRUG COMBINATIONSassays Baclofen and Torasemide + Mexiletine and Cinacalcet +Sulfisoxazole and Torasemide + Baclofen and Trimetazidine andTorasemide + Baclofen and Cinacalcet and Torasemide + Baclofen andAcamprosate and Torasemide + Sulfisoxazole and Trimetazidine andTorasemide + and Zonisamide Sulfisoxazole and Mexiletine and Torasemideand + Cinacalcet Baclofen and Acamprosate and Torasemide and +Diethylcarbamazine Baclofen and Acamprosate and Torasemide and +Ifenprodil Levosimendan and Baclofen and Trimetazidine + Levosimendanand Aminocaproic acid and + Trimetazidine Levosimendan and Terbinafineand Trimetazidine + Levosimendan and Sulfisoxazole and + Trimetazidine

IV Compounds Enhanced Behavioral and Cognitive Performances ofIntoxicated Animals

Animals are intoxicated as in the above section.

Spontaneous Alternation Performances—Y Maze Test

On day 7, all animals are tested for spontaneous alternation performancein the Y-maze, an index of spatial working memory. The Y-maze is made ofgrey polyvinylchloride. Each arm is 40 cm long, 13 cm high, 3 cm wide atthe bottom, 10 cm wide at the top, and converging at an equal angle.Each mouse is placed at the end of one arm and allowed to move freelythrough the maze during an 8 min session. The series of arm entries,including possible returns into the same arm, are checked visually. Analternation is defined as entries into all three arms on consecutiveoccasions. The number of maximum alternations is therefore the totalnumber of arm entries minus two and the percentage of alternation iscalculated as (actual alternations/maximum alternations)×100. Parametersinclude the percentage of alternation (memory index) and total number ofarm entries (exploration index). Animals that show an extreme behavior(Alternation percentage <25% or >85% or number of arm entries <10) arediscarded. Usually, it accounts for 0-5% of the animals. This testincidentally serves to analyze at the behavioral level the impact andthe amnesic effect induced in mice by the Aβ25-35 injection.

Passive Avoidance Test

The apparatus is a two-compartment (15×20×15 cm high) box with oneilluminated with white polyvinylchloride walls and the other darkenedwith black polyvinylchloride walls and a grid floor. A guillotine doorseparates each compartment. A 60 W lamp positioned 40 cm above theapparatus lights up the white compartment during the experiment.Scrambled footshocks (0.3 mA for 3 s) could be delivered to the gridfloor using a shock generator scrambler (Lafayette Instruments,Lafayette, USA). The guillotine door is initially closed during thetraining session. Each mouse is placed into the white compartment. After5 s, the door raises. When the mouse enters the darkened compartment andplaces all its paws on the grid floor, the door closes and the footshockis delivered for 3 s. The step-through latency, that is, the latencyspent to enter the darkened compartment, and the number of vocalizationsis recorded. The retention test is carried out 24 h after training. Eachmouse is placed again into the white compartment. After 5 s the doors israised, the step-through latency and the escape latency, i.e. the timespent to return into the white compartment, are recorded up to 300 s.

Positive results are observed for each for the tested combinationslisted in Table 6.

TABLE 6 Passive avoidance Y MAZE Escape Step through Drug Combinationtest latency latency Baclofen-Torasemide + + +Baclofen-Acamprosate-Torasemide + + + Mexiletine and Cinacalcet + + +Sulfisoxazole and Torasemide + + +

V Compounds of the Invention Improve Neurophysiological Concern ofNeurological Diseases

Combinations therapies are tested in the in vivo model of Aβintoxication. Their effects on several parameters which are affected inneurological diseases are assessed:

-   -   Caspases 3 and 9 expression level, considered as an indicator of        apoptosis,    -   Lipid peroxidation, considered as a marker for oxidative stress        level,    -   GFAP expression assay, considered as a marker of the level of        brain inflammation,    -   Brain Blood Barrier integrity,    -   Overall synapse integrity (synaptophysin ELISA).

Brain Blood Barrier Integrity

Experimental design about animal intoxication by AP is the same that inpart III.

The potential protective effect of the combination therapies on theblood brain barrier (BBB) integrity is analyzed in mice injectedintracerebroventricularly (i.c.v.) with oligomeric amyloid-β25-35peptide (Aβ25-35) or scrambled Aβ25-35 control peptide (Sc.Aβ), 7 daysafter injection.

On day 7 after the Aβ₂₅₋₃₅ injection, animals are tested to determinethe BBB integrity by using the EB (Evans Blue) method. EB dye is knownto bind to serum albumin after peripheral injection and has been used asa tracer for serum albumin.

EB dye (2% in saline, 4 ml/kg) is injected intraperitoneal (i.p.) 3 hprior to the transcardiac perfusion. Mice are then anesthetized withi.p. 200 μl of pre-mix ketamine 80 mg/kg, xylazine 10 mg/kg, the chestis opened. Mice are perfused transcardially with 250 ml of saline forapproximately 15 min until the fluid from the right atrium becomescolourless. After decapitation, the brain is removed and dissected outinto three regions: cerebral cortex (left+right), hippocampus (leftright), diencephalon. Then, each brain region is weighed forquantitative measurement of EB-albumin extravasation.

Samples are homogenized in phosphate-buffered saline solution and mixedby vortexing after addition of 60% trichloroacetic acid to precipitatethe protein. Samples are cooled at 4° C., and then centrifuged 30 min at10,000 g, 4° C. The supernatant is measured at 610 nm for absorbance ofEB using a spectrophotometer.

EB is quantified both as

-   -   μg/mg of brain tissue by using a standard curve, obtained by        known concentration of EB-albumin.    -   μg/mg of protein.

As mentioned in table 7, combination therapies of the invention areefficient in maintaining BBB integrity when compared with non-treatedintoxicated animals.

Overall Synapse Integrity (Synaptophysin ELISA)

Synaptophysin has been chosen as a marker of synapse integrity and isassayed using a commercial ELISA kit (USCN, Ref. E90425Mu). Samples areprepared from hippocampus tissues and homogenized in an extractionbuffer specific to as described by manufacturer and referenceliterature.

Tissues are rinsed in ice-cold PBS (0.02 mol/l, pH 7.0-7.2) to removeexcess blood thoroughly and weighed before nitrogen freezing and −80° C.storage. Tissues are cut into small pieces and homogenized in 1 mlice-cold phosphate buffer saline (PBS) solution with a glasshomogenizer. The resulting suspension is sonicated with an ultrasoniccell disrupter or subjected to two freeze-thawing cycles to furtherbreak the cell membranes. Then, homogenates are centrifugated for 5 minat 5,000 g and the supernatant is assayed immediately.

All samples are assayed in triplicates.

Quantification of proteins is performed with the Pierce BCA(bicinchoninic acid) protein assay kit (Pierce, Ref. #23327) to evaluateextraction performance and allow normalization.

The total protein concentrations are then calculated from standard curvedilutions and serve to normalize ELISA results.

Results (Table 7) show that combination therapies are efficient inmaintaining an overall Synaptophysin level in brain of treated animalswhen compared with the non-treated intoxicated animals.

Oxidative Stress Assay

Mice are sacrificed by decapitation and both hippocampi are rapidlyremoved, weighted and kept in liquid nitrogen until assayed. Afterthawing, hippocampus are homogenized in cold methanol (1/10 w/v),centrifuged at 1,000 g during 5 min and the supernatant placed ineppendorf tube. The reaction volume of each homogenate are added toFeSO4 1 mM, H2SO4 0.25 M, xylenol orange 1 mM and incubated for 30 minat room temperature. After reading the absorbance at 580 nm (A580 1), 10μl of cumene hydroperoxyde 1 mM (CHP) is added to the sample andincubated for 30 min at room temperature, to determine the maximaloxidation level. The absorbance is measured at 580 nm (A580 2). Thelevel of lipid peroxidation is determined as CHP equivalents (CHPE)according to: CHPE=A580 1/A580 2×[CHP] and expressed as CHP equivalentsper weight of tissue and as percentage of control group data.

Results (Table 7) show that combination therapies are efficient inreducing the overall oxidative stress induced by Aβ in brain of treatedanimals when compared with the non-treated intoxicated animals.

Caspase Pathway Induction Assay and GFAP Expression Assay

Mice are sacrificed by decapitation and both hippocampi are rapidlyremoved, rinsed in ice-cold PBS (0.02 mol/l, pH 7.0-7.2) to removeexcess blood thoroughly weighted and kept in liquid nitrogen untilassayed. Tissues are cut into small pieces and homogenized in 1 mlice-cold PBS with a glass homogenizer. The resulting suspension issonicated with ultrasonic cell disrupter or subjected to twofreeze-thawing cycles to further break the cell membranes. Then,homogenates are centrifugated at 5,000 g during 5 min and thesupernatant is assayed immediately.

Experiments are conducted with commercial assay: Caspase-3(USCN—E90626Mu), Caspase-9 (USCN—E90627Mu), GFAP (USCN—E90068).Quantification of proteins is performed with the Pierce BCA(bicinchoninic acid) protein assay kit (Pierce, Ref. #23227) to evaluateextraction performance and allow normalization.

Results (Table 7) show that combination therapies have a positive effecton markers of apoptosis and inflammation in brain of treated animalswhen compared with the non-treated intoxicated animals.

TABLE 7 Overall Drug Caspase Oxydative GFAP BBB Synapse Combinationpathway stress expression integrity integrity Baclofen- + + + + +Torasemide Baclofen- + + + + + Acamprosate- Torasemide Mexiletineand + + + + + Cinacalcet Sulfisoxazole and + + + + + Torasemide

B) Prevention of Glutamate Toxicity on Neuronal Cells

In this further set of experiment, candidate compounds have been testedfor their ability to prevent or reduce the toxic effects of glutamatetoxicity on neuronal cells. Glutamate toxicity is involved in thepathogenesis of neurological diseases or disorder such as MultipleSclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis,Parkinson's Disease, Huntington's Disease, neuropathies, alcoholism oralcohol withdrawal, or spinal cord injury. The drugs are first testedindividually, followed by assays of their combinatorial action.

Methods

The efficacy of drug combinations of the invention is assessed onprimary cortical neuron cells. The protocol which is used in theseassays is the same as described in section A.I.2 above.

Glutamate Toxicity Assays

The neuroprotective effect of compounds is assessed by quantification ofthe neurite network (Neurofilament immunostaining (NE)) whichspecifically reveals the glutamatergic neurons.

After 12 days of neuron culture, drugs of the candidate combinations aresolved in culture medium (+0% DMSO). Candidate combinations are thenpre-incubated with neurons for 1 hour before the Glutamate injury. Onehour after incubation with, Glutamate is added for 20 min, to a finalconcentration of 40 μM, in presence of candidate combinations, in orderto avoid further drug dilutions. At the end of the incubation, medium ischanged with medium with candidate combination but without glutamate.The culture is fixed 24 hours after glutamate injury. MK801(Dizocilpinehydrogen maleate, 77086-22-7-20 nM) is used as a positivecontrol.

After permeabilization with saponin (Sigma), cells are blocked for 2 hwith PBS containing 10% goat serum, then the cells are incubated withmouse monoclonal primary antibody against Neurofilament antibody (NF,Sigma). This antibody is revealed with Alexa Fluor 488 goat anti-mouseIgG.

Nuclei of cells are labeled by a fluorescent marker (Hoechst solution,SIGMA), and neurite network quantified. Six wells per condition are usedto assess neuronal survival in 3 different cultures.

Results

All of the tested drug combinations give a protective effect againstglutamate toxicity for cortical neuronal cells. Results are shown inTable 8 below.

As exemplified in FIGS. 24 and 25, combinations of the inventionstrongly protect neurons from glutamate toxicity under experimentalconditions described above. It is noteworthy that an effectiveprotection is noticed using drug concentrations at which drugs usedalone have no significant or lower protective effect.

TABLE 8 Neuroprotective effect against Drug Combination glutamatetoxicity Baclofen-Torasemide + Baclofen-Acamprosate-Torasemide +Mexiletine and Cinacalcet + Sulfisoxazole and Torasemide +

C) Improvement of Other Disorders Related to Glutamate ExcitoxicityUsing Combinations of the Invention

The above mentioned in vitro protective effect against glutamatetoxicity of drugs and drug combinations of the invention combined withthe protective effects exemplified herein in several AD models, promptedthe inventors to test these drugs and combinations in some models ofother diseases in the pathogenesis of which glutamate toxicity is alsoinvolved, as MS, ALS and neuropathic pain.

I) Protective Effect of Combinations in an In Vivo Model of MultipleSclerosis.

A model in which myelin-oligodendrocyte glycoprotein-immunized(MOG-immunized) mice develop chronic progressive EAE is used todemonstrate the beneficial effect of compositions of the invention inmultiple sclerosis treatment.

Animals and Chemicals

C57L/6J female mice (8 weeks old) are purchased from Janvier (France);after two weeks of habituation, female mice (10 weeks old) developchronic paralysis after immunization with MOG (Myelin OligodendrocyteGlycoprotein) peptide. The experimental encephalomyelitis is inducedwith the Hooke Kit MOG₃₅₋₅₅/CFA Emulsion PTX (Pertussis toxin) for EAEInduction (EK-0110, EK-0115; Hooke laboratories). The control kit isCK-0115 (Hooke laboratories).

Experimental Procedure

The experimental encephalomyelitis is induced by following procedure:

The day 0, two subcutaneous injections of 0.1 Ml each are performed; oneon upper back of the mouse and one in lower back. Each injectioncontains 100 μg of MOG₁₅₋₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO:1), 200 μg of inactivated Mycobacterium tuberculosis H37Ra and isemulsified in Complete Freund's adjuvant (CFA) (Hooke laboratories). Theemulsion provides antigen needed to expand and differentiateMOG-specific autoimmune T cells.

Two intraperitoneal injections of 500 ng of Pertussis toxin in PBS(Hooke kit) are performed 2 hours (Day 0) and 24 hours (Day 1) after theMUG injection. Pertussis toxin enhances EAE development by providingadditional adjuvant.

Mice develop EAE 8 days after immunization and stay chronicallyparalyzed for the duration of the experiment. After the immunization,mice are daily observed for clinical symptoms in a blind procedure.Animals are kept in a conventional pathogen-free facility and allexperiments are carried out in accordance with guidelines prescribed by,and are approved by, the standing local committee of bioethics.

Experimental Groups and Drug Treatment

Groups of female mice as disclosed are homogenized by weight before theimmunization:

-   -   Control group: vehicle injection in the same conditions of EAE        mice (from Day −1 to Day 28, placebo is given daily)    -   EAE group: MOG injection (day 0)+Pertussis toxin injections (Day        0 and 1)—from Day −1 to Day 28, placebo is given orally daily    -   EAE+positive control: MUG injection (Day 0)+Pertussis toxin        injections (Day 0 and 1)—from Day −1 to Day 28, dexamethazone is        given orally daily.    -   EAE+treatment group: MOG injection (Day 0)+Pertussis toxin        injections (Day 0 and 1). The treatments start one Day before        immunization and last until Day 28.

The clinical scores are measured at Days0-5-8-9-12-14-16-19-21-23-26-28.

Statistica software (Statsoft Inc.) is utilized throughout forstatistical analysis. ANOVA analysis and Student's t test are employedto analyse clinical disease score.P<0.05 is considered significant.

Delays of disease occurrence, clinical score and delay of death, havebeen compared between each group to the reference ‘immu’ group withKaplan-Meier curves and a Cox model (R package ‘survival’). Resultingp-values are unilateral and test the hypothesis to be better than thereference ‘immu’ group.

The total clinical score is composed of the tail score, the hind limbscore, the fore limb score and the bladder score described as below:

Tail score: Score = 0 A normal mouse holds its tail erect when moving.Score = 1 If the extremity of the tail is flaccid with a tendency tofall. Score = 2 If the tail is completely flaccid and drags on thetable.

Hind limbs score: Score = 0 A normal mouse has an energetic walk anddoesn't drag his paws Score = 1 Either one of the following tests ispositive: A - Flip test: while holding the tail between thumb and indexfinger, flip the animal on his back and observe the time it takes toright itself. A healthy mouse will turn itself immediately. A delaysuggests hind-limb weakness. B - Place the mouse on the wire cage topand observe as it crosses from one side to the other. If one or bothlimbs frequently slip between the bars we consider that there is apartial paralysis. Score = 2 Both previous tests are positive. Score = 3One or both hind limbs show signs of paralysis but some movements arepreserved; for example: the animal can grasp and hold on to theunderside of the wire cage top for a short moment before letting go.Score = 4 When both hind legs are paralyzed and the mouse drags themwhen moving.

Fore limbs score: Score = 0 A normal mouse uses its front paws activelyfor grasping and walking and holds its head erect. Score = 1 Walking ispossible but difficult due to a weakness in one or both of the paws, forexample, the front paws are considered weak when the mouse hasdifficulty grasping the underside of the wire top cage. Another sign ofweakness is head drooping. Score = 2 When one forelimb is paralyzed(impossibility to grasp and the mouse turns around the paralyzed limb).At this time the head has also lost much of its muscle tone. Score = 3Mouse cannot move, and food and water are unattainable.

Bladder score: Score = 0 A normal mouse has full control of its bladder.Score = 1 A mouse is considered incontinent when its lower body issoaked with urine.

The global score for each animal is determined by the addition of allthe above mentioned categories. The maximum score for live animals is10.

Results-Combinations Therapies are Efficient in a MS Model

A significant improvement of global clinical score is observed in “EAE+treatment group” mice, notably for the combinations listed in Table 9.

TABLE 9 Improvement of the global clinical Drug Combination score in EAEanimals Baclofen-Torasemide + Baclofen-Acamprosate-Torasemide +Mexiletine and Cinacalcet + Sulfisoxazole and Torasemide +

II. Protective Effect of Combinations in Models of ALS.

Combination therapies according to the present invention are tested invitro, in a coculture model, and in vivo, in a mouse model of ALS.Protocols and results are presented in this section.

II.1 Protective Effect Against Glutamate Toxicity in Primary Cultures ofNerve-Muscle Co-Culture Primary Cocultures of Nerve- and Muscle Cells

Human muscle is prepared according to a previously described method fromportions of biopsie of healthy patient (44). Muscle cells areestablished from dissociated cells (10000 cells per wells), plated ingelatin-coated 0.1% on 48 wells plate and grown in a proliferatingmedium consisting of mix of MEM medium and M199 medium.

Immediately after satellite cells fusion, whole transverse slices of13-day-old rat Wistar embryos spinal cords with dorsal root ganglia(DRG) attached are placed on the muscle monolayer 1 explant per well (incenter area). DRG are necessary to achieve a good ratio of innervations,innervated cultures are maintained in mix medium, After 24 h in theusual co-culture neuritis are observed growing out of the spinal cordexplants. They make contacts with myotubes and induce the firstcontractions after 8 days. Quickly thereafter, innervated muscle fibreslocated in proximity to the spinal cord explants, are virtuallycontinuously contracting. Innervated fibres are morphologically, andspatially distinct from the non-innervated ones and could easily bedistinguished from them.

One co-culture is done (6 wells per conditions).

Glutamate Injury

On day 27, co-cultures are incubated with candidate compounds orRiluzole one hour before glutamate intoxication (60 μM) for 20 min.Then, co-cultures are washed and candidate compounds or Riluzole areadded for an additional 48 h. After this incubation time, unfixedcocultures are incubated with α-bungarotoxin coupled with Alexa 488 atconcentration 500 nmol/L for 15 min at room temperature. Then,cocultures fixed by PFA for 20 min at room temperature. Afterpermeabilization with 0.1% of saponin, co-cultures are incubated withanti-neurofilament antibody (NF).

These antibodies are detected with Alexa Fluor 568 goat anti-mouse IgG(Molecular probe). Nuclei of neurons are labeled by a fluorescent marker(Hoechst solution).

Endpoints are (1) Total neurite length, (2) Number of motor units, (3)Total motor unit area, which are indicative of motorneurone survival andfunctionality. For each condition, 2×10 pictures per well are takenusing InCell Analyzer™ 1000 (GE Healthcare) with 20× magnification. Allthe images are taken in the same conditions.

Results

Drugs of the invention effectively protect motorneurones and motor unitsin the coculture model. Moreover an improvement of the protection isnoticed when drugs are used in combination for the drug combinationslisted in table 10.

TABLE 10 Protective effect against glutamate intoxication DrugCombination in muscle/nerve co-cultures Baclofen-Torasemide +Baclofen-Acamprosate-Torasemide + Mexiletine and Cinacalcet +Sulfisoxazole and Torasemide +

II.2 Combinations Therapies are Efficient in ALS Mouse Model

Experiments are performed on male mice, Transgenic male miceB6SJL-Tg(SOD1)2Gur/J mice and their control (respectively SN2726 andSN2297 from Jackson Laboratories, Ben Harbor, USA and distributed byCharles River in France) are chosen in this set of experiments to mimicALS.

Diseased mice express the SOD1-G93A transgene, designed with a mutanthuman SOD1 gene (a single amino acid substitution of glycine to alanineat codon 93) driven by its endogenous human SOD1 promoter. Control miceexpress the control human SOD1 gene.

Drug Administration

Mice are dosed with candidate drug treatment diluted in vehicle from60th day after birth till death, Diluted solutions of drug candidatesare prepared with water at room temperature just before the beginning ofthe administration.

In drinking water:

Riluzole is added in drinking water at a final concentration of 6 mg/ml(adjusted to each group mean body weight) in 5% cyclodextrin. As a mousedrinks about 5 ml/day, the estimated administrated dose is 30 mg/kg/daywhich is a dose that was shown to increase the survival of mice.

-   -   Cyclodextrine is used as vehicle at the final concentration of        5%, diluted in water at room temperature from stock solution        (cyclodextrin 20%).

Oral administration (per os):

-   -   Drug combinations are administrated per os, daily.    -   Cyclodextrine is used as vehicle at the final concentration of        5%, diluted in water at room temperature from stock solution        (cyclodextrin 20%).

Clinical Observation

The clinical observation of each mouse is performed daily, from thefirst day of treatment (60 days of age) until the death (or sacrifice).Clinical observation consists in studying behavioural tests: onset ofparalysis, “loss of splay”, “loss of righting reflex”, and general gaitobservation:

-   -   Onset of paralysis: The observation consists of paralysis        observation of each limb. Onset of paralysis corresponds to the        day of the first signs of paralysis.    -   The loss of splay test consists of tremors or shaking        notification and the position of hind limb (hanging or splaying        out) when the mouse is suspended by the tail.    -   The loss of righting reflex test evaluates the ability of the        mouse to right itself within 30 sec of being turned on either        side. The righting reflex is lost when the mouse is unable to        right itself. The loss of righting reflex determines the end        stage of disease: the mouse unable to right itself is        euthanized.

Results-Combinations Therapies are Efficient in ALS In Vivo Model

An improvement of the disease is observed for the diseased animalstreated with the drugs and drug combinations of the invention. Notably,drugs combinations listed in Table 11 efficiently improve clinical scoreof these animals during the different stage of the disease.

TABLE 11 Effect on clinical score in Drug Combination diseased animalsBaclofen-Torasemide + Baclofen-Acamprosate-Torasemide + Mexiletine andCinacalcet + Sulfisoxazole and Torasemide +

III) Protective Effect of Combinations of the Invention in OxaliplatinInduced Neuropathy as an In Vivo Model for Neuropathic Pain.

Combinatorial therapies of the present invention are tested in vivo, insuitable models of peripheral neuropathy, i.e., acute model ofoxaliplatin-induced neuropathy and chronic model of oxaliplatin-inducedneuropathy. The animals, protocols and results are presented in thissection.

Animal Husbandry

Sprague-Dawley rats (CERJ, France), weighing 150-175 g at the beginningof the experimental of the Oxaliplatin treatment (D₀) are used. Animalsare housed in a limited access animal facility in a temperature (19.5°C.-24.5° C.) and relative humidity, (45%-65%) controlled room with a 12h-light/dark cycle, with ad libitum access to standard pelletedlaboratory chow and water throughout the study. Animals are housed 4 or5 per cage and a one week-acclimation period is observed before anytesting.

Experimental Design

Four following groups of rats are used in all experiments:

Control Groups:

Group 1: Vehicle of Oxaliplatin (distilled water), i.p./Vehicle ofcandidate combination(s) (Distilled water), p.o. daily.

Group 2: Oxaliplatin (distilled water), i.p./Vehicle of candidatecombination(s) (Distilled water), p.o. daily.

Group 3: Oxaliplatin 3 mg/kg i.p./single drug in Distilled water, p.o.daily×9.

Tested Composition Groups:

Group 4: Oxaliplatin 3 mg/kg i.p./candidate combination(s) in Distilledwater, p.o. daily×9.

Group 5: Oxaliplatin 3 mg/kg i.p./Gabapentin (100 mg/kg) in Distilledwater, p.o. on testing days (i.e. D₁ & D₈);

Vehicle and test items are delivered daily from D−1 to D7 (the daybefore the last testing day) whereas Gabapentin is administered ontesting days (120 minutes before the test).

All treatments are administered in a coded and random order when it ispossible. Doses are expressed in terms of free active substance.

Neuropathy Induction

Acute neuropathy is induced by a single intraperitoneal injection ofoxaliplatin (3 mg/kg).

Chronic peripheral neuropathy is induced by repeated intraperitonealinjections of oxaliplatin (3 mg/kg, i.p.) on days 0, 2, 4 and 7 (CD=12mg/kg, i.p.). Chronic neuropathy in humans is cumulative as well and ismost commonly seen in patients who have received total doses ofoxaliplatin > or =540 mg/m² which corresponds to ˜15 mg/kg as cumulativedose in rats (Cersosimo R. J. 2005).

The oxaliplatin-induced painful neuropathy in rat reproduces the painsymptoms in oxaliplatin-treated patients:

-   -   The thermal hyperalgesia is the earliest symptom. It can be        measured with the acetone test or with the tail-immersion test;    -   The mechanical hyperalgesia appears later. It can be quantified        with the Von Frey test or the paw pressure test.

Animal Dosing and Testing

All drug combinations are administered from the day before the firstintraperitoneal injection of oxaliplatin 3 mg/kg (D−1) and pursued dailyorally until D7. During the testing days (i.e. D1 and D7), the drugcombinations are administered after the test. Animals from thereference-treated group (gabapentin) are dosed only during the testingdays.

Acetone Test

Cold allodynia is assessed using the acetone test by measuring theresponses to thermal non-nociceptive stimulation on D1 (around 24 hafter the first injection of oxaliplatin 3 mg/kg (acute effect ofoxaliplatin), and D8 (chronic effect of oxaliplatin). In the acetonetest, latency of hindpaw withdrawal is measured after application of adrop of acetone to the plantar surface of both hindpaws (reaction time)and the intensity of the response is scored (cold score). Reaction timeto the cooling effect of acetone is measured within 20 sec (cut-off)after acetone application. Responses to acetone are also graded to thefollowing 4-point scale: 0 (no response); 1 (quick withdrawal, flick ofthe paw); 2 (prolonged withdrawal or marked flicking of the paw); 3(repeated flicking of the paw with licking or biting).

For each experimental group, results are expressed as:

-   -   The reaction time defined as the time expressed in sec required        to elicit paw reaction (mean of 6 measures for each rat together        ±SEM).    -   The cumulative cold score defined as the sum of the 6 scores for        each rat together ±SEM. The minimum score being 0 (no response        to any of the 6 trials) and the maximum possible score being 18        (repeated flicking and licking or biting of paws on each of the        six trials).

Statistical Analyses

Student test, unilateral, type 3 is performed. The significance level isset as p<0.05; all the groups are compared to the diseased-L vehiclegroup (oxaliplatin treated group). Means and standard error mean areshown on the figures.

Results

Oxaliplatin induced a significant decrease in reaction time of pawwithdrawal after acetone application (diseased group+vehicle) during thetime course. This decrease is progressive and significant from day 1(acute model of oxaliplatin-induced neuropathy) to day 8 (chronic model)as compared to the vehicle group.

Anti-Allodynic Effect in Acute Model of Oxaliplatin-Induced Neuropathy

The drug combinations tested in acute model of oxaliplatin-inducedneuropathy, are assessed with acetone test. Table 12 presents drugcombinations (Group 4) which induce a significant decrease in thecumulative cold score and a significant increase of reaction time ascompared to the oxaliplatin-vehicle treated group (Group 2). Inconclusion, these drug combinations protect animals from acuteneuropathy induced by oxaliplatin.

TABLE 12 Variation of Drug combinations tested the cold score Reactiontime Anti- in acute model of compared to compared to allodynicneuropathy (Group 4) Group 2 Group 2 effect Baclofen-Torasemide decreaseincrease + Baclofen-Acamprosate- decrease increase + TorasemideMexiletine and decrease increase + Cinacalcet Sulfisoxazole and decreaseincrease + Torasemide + = anti-allodynic effect obtained in Group 4 ofrats, following analysis of the cumulative cold scores and analysis ofthe reaction time in acetone tests, in acute oxaliplatin-induced model.

Anti-Allodynic Effect in Chronic Model of Oxaliplatin-Induced Neuropathy

The drug combinations used in chronic model of oxaliplatin-inducedneuropathy are assessed with acetone test.

Table 13 presents drug combinations, for which, the reaction time andthe cold score in acetone test measured in the Group 4 (animals treatedwith drug combinations and oxaliplatin) are respectively significantlyincreased and decreased after the treatment in chronic model ofneuropathy compared to the oxaliplatin-vehicle treated group (Group 2).In conclusion, these drug combinations protect animals from chronicneuropathy induced by oxaliplatin.

TABLE 13 Variation of Drug combinations tested the cold score Reactiontime Anti- in Chronic model of compared to compared to allodynicneuropathy (Group 4) Group 2 Group 2 effect Baclofen-Torasemide decreaseincrease + Baclofen-Acamprosate- decrease increase + TorasemideMexiletine and decrease increase + Cinacalcet Sulfisoxazole and decreaseincrease + Torasemide + = anti-allodynic effect obtained in Group 4 ofrats, following analysis of the cumulative cold scores and analysis ofthe reaction time in acetone tests, in chronic oxaliplatin-inducedmodel.

IV) Baclofen-Torasemide Based Compositions Promote Nerve Regeneration inNon Intoxicated Cells

Neurotrophic (Weds of Baclofen Torasemide Combination In Vitro NeuriteLength Assay

Neurite growth evaluation within 10 days old cultures of rat corticalcells was performed using MAP-2 antibodies as mentioned in section A)11.4, with the exception that cells have not been exposed to any toxic.10 days old cell cultures were incubated with the drugs for 1 day beforethe assay.

Results

As shown in FIG. 28, baclofen-torasemide combination exhibits asignificant neurotrophic effect (+11%) whereas individual drugs, whenused alone, do not have any substantial neurotrophic effect. Indeed, asignificant increase in total neurite length within neuronal network(MAP2-2 labelling) is observed upon exposure to baclofen-torasemidecombination (400 nM and 80 nM respectively). Noteworthy, neithercombination nor drugs alone have an effect on the number of neurones,thereby stressing that this increase in neurite network is related to anextension of existing neurites and to the promotion of de novo neuronalcell extensions.

These results confirm that baclofen-torasemide combination is efficientin supporting axon extension and thus is efficient in treating spinalcord injury and others nerves injuries. It confirms also that thecombination is efficient in treating inherited neuropathies comprisingeither an axonal, demyelinating, or both axonal and demyelinatingcomponent. Indeed, it should be considered that demyelination causes adestabilisation of the axon which results in axonal degenerationobserved even in the neuropathies considered to be mainly of thedemyelinating form.

Baclofen-Torasemide Based Compositions are Efficient in Promoting NerveRegeneration In Vivo

Sciatic nerve crush is widely accepted as a valid model for peripheralnerve injury and for the assessment of nerve regeneration. In thismodel, nerve damage results in rapid disruption of nerve function asevidenced by the measure of the evoked muscle action potential (CMAPs)generated through the stimulation of the injured sciatic nerve.

Nerve injury is characterized by a lower nerve conduction of the signalthat results by an increased latency in generation of CMAP and by animpaired strength of action potential resulting in a decreased amplitudeand duration.

Usually, first signs of recovery of nerve function occur within 2 weeks,and, by week 4 post-lesioning, a significant remyelination of theregenerated axons is observed in the sciatic nerve by histology (45).

Nerve Crush

Mice were anesthetized using isoflurane (2.5 to 3% in air). The rightthigh was then shaved and the sciatic nerve exposed at mid-thigh leveland crushed at 5 mm proximal to the bifurcation of the sciatic nerve.The nerve were crushed for 10 s twice with a microforceps (Holtex,reference P35311) with a 90″ rotation between each crush. For shamoperated animals, sciatic nerves were exposed but not crushed. Finally,the skin incision was secured with wound clips. The day of the crush isconsidered as day 0.

Dosage Schedule

The day of the crush, first administration of compounds was performed 30min after the crush.

Compounds are then after administered twice daily, from the day afterthe crush and during 42 days. Within a single day, drug administrationswere spaced by at least 6 hours.

On test days (days 7 and 30) mice were administered 1 h30 before thetest. The volume of administration was 10 ml/kg, in 0.25% DMSO/sterilewater.

Dose 1 (bid) Dose 2 (bid) Dose 3 (bid) Baclofen (+/−) 3 mg/kg 3 mg/kg 3mg/kg Torasemide 25 μg/kg 100 μg/kg 400 μg/kg

Electromyography Measures

Electrophysiological recordings were performed using a Keypointelectromyography (EMG) (Medtronic, France) on Day 7 and Day 30. Micewere anaesthetized by 2.5-3% isoflurane in air. Subcutaneous monopolarneedle electrodes were used for both stimulation and recording.Supramaximal (12.8 ma) square waves pulses of 0.2 ins duration were usedto stimulate the sciatic nerve. The right sciatic nerve (ipsilateral)was stimulated with single pulse applied at the sciatic notch. CMAP wasrecorded by needle electrodes placed at the gastrocnemius muscle. Theonset (latency) of CMAP signal expressed in milliseconds is used toestimate the nerve conduction velocity. Latency thus reflects the degreeof myelinisation of the axons. The amplitude of the action (μV)potential was also determined, it reflects the level of denervation andof reinervation of muscles. Amplitude of CHAP is currently given asproportional to the number of regenerated motor axons. Duration of theevoked muscle potential was also determined. Amplitude and duration ofthe evoked muscle potential are more related to muscle reinervation.Latency and Amplitude are more generally recognized as the mostimportant endpoints when dealing with nerve regeneration. Data wereanalyzed with a bilateral, type 3, Student t-test; significant level isset at p<0.05.

Results

Baclofen-torasemide combinations are efficient, at all the tested doses,in significantly improving time latency of signal when compared withvehicle treated animals, and this as soon a the seventh day from thenerve injury (FIG. 29, A). A significant difference is still observed atthe 30^(th) day from the nerve crush, when usually a beginning for aspontaneous recovery is observed.

A statistically significant improvement in the amplitude of the signalis also observed for the Dose 3 on the 30^(th) day from the nerve crush(FIG. 29, B). Such an improvement is also observed, but to a lesserextent, for doses 1 and 2. Similar results are observed when measuringthe duration of the signal.

Altogether, these in vivo results show the efficiency ofbaclofen-torasemide combination in promoting nerve regeneration throughremyelinisation and muscle reinervation. Hence in vim experimental dataconfirm the neurotrophic effects of baclofen-torasemide observed invitro and their usefulness in correcting neuropathies where nerves ofthe peripheral nervous system are damaged (i.e. neuropathies as definedin the specification) but also spinal cord injury.

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1-13. (canceled)
 14. A method of treating Parkinson's disease in asubject in need thereof, comprising administering to said subject aneffective amount of torasemide and baclofen, or prodrugs, or salts, orsustained release formulations thereof.
 15. The method of claim 14,further comprising administering to said subject a compound selectedfrom the group consisting of acamprosate, mexiletine, sulfisoxazole,cinacalcet and tadalafil, or prodrugs, or salts, or sustained releaseformulations thereof.
 16. The method of claim 14, wherein the compoundsare administered with a pharmaceutically acceptable carrier orexcipient.
 17. The method of claim 14, wherein the compounds areformulated or administered together, separately or sequentially.
 18. Themethod of claim 14, wherein the compounds are administered repeatedly tothe subject.
 19. The method of claim 14, wherein the compounds areadministered orally.
 20. The method of claim 14, wherein torasemide isadministered in a dosage from about 0.05 to 4 mg per day.
 21. The methodof claim 14, wherein baclofen is administered in a dosage from about0.15 to 15 mg per day.